Nanoparticles and nanoparticle compositions

ABSTRACT

The invention provides multivalent surface-crosslinked micelle (SCM) particles, crosslinked reverse micelle (CRM) particles, and methods of making and using them. The SCM particles can be used, for example, to inhibit a virus or bacteria from binding to a host cell. The inhibition can be used in therapy for the flu, cancer, or AIDS. The CRM particles can be used, for example, to prepare metal nanoparticles or metal alloy nanoparticles, or they can be used in catalytic reactions.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.14/316,585, filed Jun. 26, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/640,698, filed Dec. 18, 2012, which is anationalization under 35 U.S.C. 371 of PCT/US2011/031736, filed 8 Apr.2011 and published as WO 2011/130114 A1 on 20 Oct. 2011, which claimsthe benefit of U.S. Provisional Application Ser. No. 61/323,072, filed12 Apr. 2010, entitled “Nanoparticles and Nanoparticle Compositions”which application are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under CHE0748616 awardedby The National Science Foundation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

This invention relates stable nanoparticles and nanoparticlecompositions. The nanoparticles and nanoparticle compositions areprepared using surface crosslinked micelles (SCM) and are capable ofincorporating materials such as pharmaceuticals and metal atoms.

BACKGROUND

With more than 12 million new cases of cancer each year, development ofnew and more effective anticancer therapies is a major priority ofpharmaceutical researchers. Targeted delivery of therapeutic agents isparticularly important in chemotherapy because anticancer drugs tend tohave severe side effects, and anticancer drugs often kill healthy cells.

Both passive and active targeting have been employed in the delivery ofanticancer drugs. Passive targeting relies on the enhanced permeabilityand retention (EPR) effect of cancerous tissues, a result of leaky bloodvessels and poor lymphatic drainage. Active targeting depends onfunctionalizing the surface of the drug carrier with molecules that bindto overexpressed receptors on cancerous cells. Despite the benefits ofactive and passive targeting, compositions and methods with increasedeffectiveness are needed to further improve current cancer therapy.

Multivalent interactions frequently occur between biological entities.When strong binding is not possible with a single receptor-ligand pair,multivalency, or simultaneous binding between multiple receptors andligands, can be an effective strategy to enhance the binding.Multivalent interactions are involved in many diseases, such as thecommon flu, cancer, and AIDS. Researchers have sought to developmultivalent ligands that can inhibit the binding of host cells byviruses and bacteria. Two of the most widely used scaffolds inmultivalency are dendrimers and gold nanoparticles protected withmultiple functionalized thiols. However, few clinical applications havebeen developed from these approaches.

Accordingly, there is a need for improved therapies for conditions thatinvolve multivalent interactions. Multivalent ligands that can inhibitthe binding of host cells by viruses, bacteria, and the like, are neededfor such therapy. There is also a need for improved methods to preparemultivalent ligands, such as multivalent ligands in the form ofnanoparticles.

SUMMARY

The invention provides stable nanoparticles and methods for the facilepreparation of such nanoparticles. New methods to crosslink surfactantsto afford multivalent organic nanoparticles are described herein.Micelles and reversed micelles can be prepared from various surfactants,and the micelles can be surface functionalized. The micellenanoparticles can be either water soluble, or organic-soluble, and theycan encapsulate “cargo” molecules that can be released upon exposure tospecific stimuli. The nanoparticles can be used for a variety ofpurposes, including biomedical and chemical applications. Thenanoparticles can be used as drug delivery vehicles, for example, byencapsulating suitable drugs. The drugs can be released at, for example,the site of an inflammation or a tumor.

The invention also provides new scaffolds for multivalent ligands.Water-soluble organic nanoparticles were prepared by crosslinkingalkyne-containing surfactant micelles using highly efficient “click”reactions. Tens of ligands were attached to surface-crosslinked micelles(SCMs). Hydrophobic guests can be encapsulated inside the SCMs. Whenreversible crosslinkers are used, the hydrophobic guest molecules can bereleased upon cleavage of the crosslinkers, making the SCMs suitable foruse as drug-delivery systems. The micelles can be prepared fromsurfactants that can be synthesized in a few simple steps from readilyavailable and inexpensive starting materials. The self-assemblingapproach, crosslinking strategy, and post-modification employed inpreparing the SCMs can also be used to prepare organic-solublecrosslinked reversed micelles (CRMs).

Accordingly, the invention provides an organic particle comprisingsurface crosslinked non-polymeric organic amphiphiles, wherein polarhead groups of the amphiphiles are covalently crosslinked to each otherat the surface of the particle through triazole groups or thioethersgroups, and tail groups of the amphiphiles are arranged toward theinterior of the particle: and the particle is water-soluble. Theparticle can include one or more cargo molecules, such as drugmolecules, within the particle or at the surface of the particle. Theinvention also provides a delivery system comprising a plurality ofparticles described herein, and a pharmaceutically acceptable diluent orcarrier.

The invention further provides an organic particle comprisingnon-polymeric crosslinked amphiphiles; where the amphiphiles compriseone or more nonpolar alkyl or fluoroalkyl chains and one or more polarhead groups; the nonpolar chains are located on the exterior of theparticle and the polar head groups are oriented toward the interior ofthe particle; and the amphiphiles are covalently crosslinked to eachother near the head groups through triazole groups or thioether groups.These particles can be crosslinked reverse micelle (CRM) particles, andthe particles can include one or more metal salts or metal particles inthe core of the particle.

The invention also provides methods for preparing a surface-crosslinkedorganic particle. The method can include (a) combining a plurality ofnon-polymeric amphiphiles and water to form a noncovalently associatedself-assembled micellar structure; where the non-polymeric amphiphileshave polar head groups and non-polar tail groups, and the polar headgroups comprise two or more alkynyl groups or azido groups; (b)combining the self-assembled structure with a plurality of crosslinkingagents, wherein the crosslinking agents comprise two or more azidogroups or two or more alkynyl groups; and (c) inducing cycloadditionbetween the alkynes and azides, thermally or with a suitable catalyst,to covalently crosslink the amphiphiles to each other near the headgroups through formation of triazole groups.

In preparing the micelles, the amphiphiles can self-assemble throughhydrophobic interactions among the hydrophobic groups. The particles canbe formed in the presence of cargo molecules so that the amphiphilesencapsulate the cargo molecules. The cargo can be, for example, one ormore drugs, organic nanoparticles, inorganic nanoparticles,fluorophores, diagnostic agents, catalysts, or a combination thereof.The surface of the crosslinked particles can readily be functionalizedusing click chemistry reactions. For example, the surface-crosslinkedparticle can be contacted with one or more azido-containing oralkynyl-containing compounds such as water-soluble polymers,fluorophores, biological ligands, nucleic acids or analogues thereof, ora combination thereof; followed by inducing cycloaddition betweenalkynes or azides on the surface of the particle with theazido-containing or alkynyl-containing compounds, where thecycloaddition is induced thermally or with a suitable catalyst; toprovide a water soluble multivalent particle that has a plurality ofwater-soluble polymers, fluorophores, biological ligands, nucleic acidsor analogues thereof, or a combination thereof, linked to the surface ofthe particle through triazole groups.

The invention additionally provides a method for preparing asurface-crosslinked particle that includes (a) combining a plurality ofnon-polymeric amphiphiles and water to form a noncovalently associatedself-assembled structure; where the non-polymeric amphiphiles have polarhead groups and non-polar tail groups, and the polar head groupscomprise two or more alkenyl groups; (b) combining the self-assembledstructure with a plurality of crosslinking agents, wherein thecrosslinking agents comprise two or more thiol groups; and (c) inducingthiol-ene addition between the alkenes of the amphiphiles and the thiolgroups of the crosslinkers photochemically to covalently crosslink theamphiphiles to each other near the head groups through the formation ofthioether groups.

The invention further provides a method for preparing an organicparticle that includes (a) combining a plurality of non-polymericamphiphiles, water, and one or more nonpolar organic solvents, where theamphiphiles comprise one or more alkyl or fluoroalkyl chains and one ormore polar head groups, to provide a noncovalently associatedself-assembled structure; where the amphiphiles comprise two or morealkenyl groups near the head group of the amphiphile, the alkyl orfluoroalkyl chains of the amphiphiles are oriented on the exterior ofthe self-assembled structure, and the polar head groups are orientedtoward the interior of the self-assembled structure; and (b) irradiatingthe self-assembled structures, in the presence of a plurality ofcrosslinking agents comprising two or more thiol groups, and aphotoinitiator, to induce crosslinking at the interior of the structure;to provide an organic particle comprising amphiphilic moietiescrosslinked by thioether groups.

The invention also provides a method of forming a metal nanoparticlecomprising (a) contacting a metal salt and a plurality of particlesdescribed herein, for example, those that have nonpolar chains locatedon the exterior of the particle and polar head groups oriented towardthe interior of the particle, in an aqueous/organic solvent mixture,thereby extracting metal ions of the metal salt into the organicsolvent, wherein the metal ions migrate to the interior of the particle,to provide a crosslinked organic particle encapsulating metal ions; and(b) contacting the crosslinked organic particle encapsulating metal ionswith a reducing agent, thereby reducing the metal ions in the interiorof the crosslinked organic particle, to provide the metal nanoparticle.

The invention yet further provides a therapeutic method comprisingadministering to a patient in need of therapy an effective amount of thedelivery system described herein, where the surface crosslinking of theparticles encapsulate one or more drugs, the surface crosslinking of theparticles is cleaved in vivo, and the drug of the particles is releasedinto the body of the patient, thereby providing the drug to the patient.

The invention thus provides for the use of the compositions describedherein for medical therapy. The medical therapy can be, for example,treating inflammation, treating a viral infection, treating a bacterialinfection, or treating cancer, such as breast cancer, lung cancer,pancreatic cancer, or colon cancer. The invention also provided for theuse of a composition described herein for the manufacture of amedicament to treat such conditions. The medicament can include apharmaceutically acceptable diluent, excipient, or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention, however, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1 illustrates the preparation of Surface Crosslinked Micelles(SCMs) and post-functionalization by click chemistry, according to anembodiment.

FIG. 2 illustrates the preparation of Crosslinked Reversed Micelles(CRMs) and templated synthesis of gold nanoparticles, according to anembodiment.

FIG. 3 illustrates preparation of surface-crosslinked camptothecinnanoparticles by precipitation and click reaction, according to anembodiment.

FIG. 4 illustrates post-functionalization of water-solublenanoparticles, catalyzed by a Cu(I) catalyst in solution, by addition ofazide-functionalized polymers or ligands, according to an embodiment.The azide-functionalized polymers or ligands can be, for example,compounds 4, 5, or 6, illustrated in FIG. 1, compounds 12 or 13,illustrated in FIG. 3, or a combination thereof.

FIG. 5 illustrates ¹H NMR spectra of a 10 mM micellar solution of 1: (a)in D₂O, (b) after addition of 1 equiv of 2a, (c) after crosslinking, and(d) after dialysis to remove water-soluble impurities.

FIG. 6 illustrates a ¹H NMR spectrum of PEG-SCMs after treatment with anexcess of HIO₄, overnight at room temperature.

FIG. 7 illustrates a TEM micrograph of PEG-SCMs, stained with 2%phosphotungstic acid aqueous solution.

FIG. 8 illustrates ¹H NMR spectra of a 2:3 mixture of 10 and 2 ofExample 2: (a) before irradiation, (b) after UV irradiation for 10hours, and (c) after washing with water.

FIGS. 9A-9F illustrate the hydrodynamic radii of aggregates of CRMsdetermined by DLS in (a) acetone at 0 min, (b) acetone at 40 min, (c)butanone, (d) chloroform, and (e) THF before mass-normalization and (f)THF after mass-normalization.

FIGS. 10A-10B illustrate TEM micrographs of (a) unstained CRMs and (b)CRMs stained with 2% phosphotungstic acid.

FIG. 11 illustrates UV-vis spectra of Au-CRMs prepared with [AuCl₄⁻]/[10]=1 (line W), 0.1 (line X), and 0.02 (line Y), according to theprocedures described in Example 2. The dotted spectral line is that of[AuCl₄ ⁻]/[10]=0.1 multiplied by 10. [10]=5×10⁻⁴ M.

FIGS. 12 and 13 illustrate the templated synthesis of metalnanoparticles, according to various embodiments, including firstextracting AuCl₄ ⁻ (FIG. 12) or the mixture of AuCl₄ ⁻ and PtCl₆ ²⁻(FIG. 13) into organic solvents, followed by reduction (e.g., with NaBH₄or other reducing agents); the gold nanoparticles or Au—Pt alloy may befluorescent, and can be characterized by DLS, fluorescence, or TEM.

FIG. 14 illustrates normalized emission spectra of pyrene in thepresence of surfactant 1 in water.

FIG. 15 illustrates pyrene I₃/I₁ ratio as a function of theconcentration of surfactant 1.

FIG. 16 illustrates the change of the pyrene I₃/I₁ ratio after additionof 0 (Δ), 1 (□), 10 (⋄), and 100 (x) equivalents of cleaving agent topyrene-containing SRMs. Crosslinker=2, cleaving agent=HIO₄.

FIG. 17 illustrates the change of the pyrene I₃/I₁ ratio after additionof 0 (Δ), 1 (□), 10 (⋄), and 100 (x) equivalents of cleaving agent topyrene-containing SRMs. Crosslinker=3, cleaving agent=5; [1]=2×10⁻⁵ M.

FIG. 18 illustrates the relative intensity of scattered light for theSRMs upon different stimulation. Stimulation was 1 equivalents of HIO₄for SRMs crosslinked with 2 (Δ), 1 equivalents of acetal 4 for SRMscrosslinked with diazide 3 (□), and pH 5 (⋄) and pH 7 (x) acetate bufferat 37° C. for SRMs crosslinked with dithiol 5.

FIG. 19 illustrates the preparation of an SCM-encapsulatedphosphine-rhodium catalyst.

FIG. 20 illustrates the release of carboxyfluoroscein (CF) fromosmotically stressed SC-LUV

FIG. 21 illustrates the preparation of alkynyl-SCM.

FIG. 22 illustrates the preparation of the pyrene-containing SCM

FIG. 23 illustrates the preparation of the alkynyl-SCMs

DETAILED DESCRIPTION

About half of potential drug candidates identified in high throughputscreening have poor water solubility. These potential drug candidatesare often denied further chance of development because of suchsolubility problems. Although surfactant micelles can solubilizehydrophobic agents in water, their use is drug delivery is oftenhampered by high critical micelle concentration (CMC), low thermodynamicstability, and the exceedingly dynamic nature of the assembly.

Polymeric micelles represent significant improvements over surfactantmicelles because macromolecular amphiphiles tend to aggregate atconcentrations orders of magnitude lower than their small moleculecounterparts and they produce micelles with greater thermodynamicstability. A hydrophobic drug may be physically trapped inside thehydrophobic core of a polymeric micelle or covalently attached to themacromolecular amphiphile. The latter approach enables controlledrelease of drugs by specific stimuli and is more effective at preventingpremature drug-release than physical entrapment-features of particularimportance in the delivery of drugs with high cytotoxicity. It has beenreported, for example, that physically entrapped anticancer drugsdisplay as high cytotoxicity as the small molecule versions (Y. Bae, K.Kataoka, Adv. Drug. Deliv. Rev. 2009, 61, 768). Nonetheless, covalentlinking between the drug and the delivery vehicle puts significantconstraints on the structure of both components and adds considerablecomplexity to the production and formulation of the therapeutic package.

Disclosed herein is a simple method to capture the micelles ofalkynylated surfactants such as amphiphile 1 by covalent crosslinking.Crosslinking is readily achieved by the highly efficient alkyne-azideclick reaction in the presence of 1 equiv 2 and a catalytic amount ofCu(I). The alkyne-azide click reaction was well described by Rostovtsev,Green, Fokin, and Sharpless (Angew. Chem. Int. Ed. 2002, 41, 2596), aswell as Tomoe, Christensen, and Meldal (J. Org. Chem. 2002, 67, 3057).Sodium ascorbate (25 mol %) and CuCl₂ (5 mol %) and were found to workwell in the reaction mixture to facilitate the crosslinking. Aftercovalently crosslinking amphiphile 1, the resulting surface-crosslinkedmicelles (SCMs), typically 8-10 nm in diameter, have numerous residualalkynes on the surface. Multivalent post-modification can be readilyaccomplished by the same click reaction by adding desiredazide-functionalized polymers or ligands after the crosslinking.

Accordingly, the invention provides multivalent surface-crosslinkedmicelle (SCM) particles, as well as crosslinked reverse micelle (CRM)particle, and methods of making and using them. The SCMs can be used to,for example, inhibit a virus or bacteria from binding to a host cell,such as in therapy used to treat the flu, cancer, or AIDS. The CRMs canbe used, for example, to prepare metal nanoparticles or metal alloynanoparticles.

Definitions

As used herein, the recited terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 14^(th) Edition, by R. J. Lewis,John Wiley & Sons, New York, N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a compound” includes a plurality of such compounds, so that acompound X includes a plurality of compounds X It is further noted thatthe claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” and the like, inconnection with the recitation of claim elements or use of a “negative”limitation.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrase “one or more” is readily understood by one of skill in the art,particularly when read in context of its usage. For example, one or moresubstituents on a phenyl ring refers to one to five, or one to four, forexample if the phenyl ring is disubstituted.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer. Unless indicated otherwise herein, the term“about” is intended to include values, e.g., weight percents, proximateto the recited range that are equivalent in terms of the functionalityof the individual ingredient, the composition, or the embodiment.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible subranges andcombinations of subranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percents or carbon groups) includes each specific value, integer,decimal, or identity within the range. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths, ortenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than,”“or more,” and the like, include the number recited and such terms referto ranges that can be subsequently broken down into subranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all subratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, theinvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, the invention encompassesnot only the main group, but also the main group absent one or more ofthe group members. The invention therefore envisages the explicitexclusion of any one or more of members of a recited group. Accordingly,provisos may apply to any of the disclosed categories or embodimentswhereby any one or more of the recited elements, species, orembodiments, may be excluded from such categories or embodiments, forexample, as used in an explicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vive.

An “effective amount” refers to an amount effective to treat a disease,disorder, and/or condition, or to bring about a recited effect. Forexample, an amount effective can be an amount effective to reduce theprogression or severity of the condition or symptoms being treated.Determination of a therapeutically effective amount is well within thecapacity of persons skilled in the art. The term “effective amount” isintended to include an amount of a micelle composition described herein,or an amount of a combination of micelles described herein, e.g., thatis effective to treat or prevent a disease or disorder, or to treat thesymptoms of the disease or disorder, in a host. Thus, an “effectiveamount” generally means an amount that provides the desired effect.

The terms “treating”, “treat” and “treatment” include (i) preventing adisease, pathologic or medical condition from occurring (e.g.,prophylaxis); (ii) inhibiting the disease, pathologic or medicalcondition or arresting its development; (iii) relieving the disease,pathologic or medical condition; and/or (iv) diminishing symptomsassociated with the disease, pathologic or medical condition. Thus, theterms “treat”, “treatment”, and “treating” extend to prophylaxis andinclude prevent, prevention, preventing, lowering, stopping or reversingthe progression or severity of the condition or symptoms being treated.As such, the term “treatment” includes medical, therapeutic, and/orprophylactic administration, as appropriate.

The terms “inhibit”, “inhibiting”, and “inhibition” refer to theslowing, halting, or reversing the growth or progression of a disease,infection, condition, or group of cells. The inhibition can be greaterthan about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for example, comparedto the growth or progression that occurs in the absence of the treatmentor contacting.

The term “multivalent” refers to the capacity of a ligand, e.g., amicelle, to simultaneous bind multiple receptors.

The term “drug” refers to a therapeutic organic compound that can treata disease or condition. Examples of drugs suitable for encapsulation inthe micelles or liposomes described herein include doxorubicin,daunorubicin, vincristine, paclitaxel, neocarzinostatin, calicheamicin,cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin,lurtotecan, annamycin, docetaxel, tamoxifen, epirubicin, methotrexate,vinblastin, vincristin, topotecan, and nucleic acids such as DNA, RNA,small interfering RNA (siRNA), or their analogues. An example of awater-insoluble drug is camptothecin. An example of a water-soluble drugis doxorubicin. The classification of drugs as hydrophobic or watersoluble is well known to those of skill in the art.

The term “diagnostic agent” refers to a compound that can be used to aidthe presence, location, or severity of a condition in a cell, tissue, orpatient. Examples of diagnostic agents suitable for encapsulation in themicelles, vesicles or liposomes described herein include quantum dots,gadolinium-(III) complexes for magnetic resonance imaging (MRI),magnetic nanoparticles, gold or silver nanoclusters, and organicfluorophores.

As used herein, term “crosslinked amphiphile” refers to an amphiphilethat has an alkyne or alkene group, or alternatively, an azido or thiogroup, that has undergone a click reaction with a corresponding anazido, thio, alkyne, or alkene group. Non-limiting examples ofamphiphiles suitable for crosslinking are illustrated in Schemes 5,7-10, and 12-14.

As used herein, the term “crosslinking moiety” refers to adi-functionalized group that can, by use of a click reaction, covalentlybond to an appropriately functionalized amphiphile. Non-limitingexamples of crosslinking agents, or “crosslinkers”, suitable forcrosslinking with amphiphiles are illustrated in Schemes 6 and 11.

The term “non-polar hydrocarbon or fluorocarbon tail group” refers to astraight or branched alkyl chain that is attached to a head group of anamphiphile. The hydrocarbon or fluorocarbon tail can include about 6 toabout 50 carbon atoms, or the tail can have a carbon atoms total of anyinteger between about 6 and about 20. Other chain lengths can be used,as described below. The non-polar hydrocarbon or fluorocarbon tail groupcan be any length of carbons that allows for the amphiphile to selfassemble to form micelles.

The term “non-polymeric organic amphiphiles” refers to any amphiphilethat does not include repeating monomer units, such as in polypeptides,to form the amphiphile. The term also excludes amphiphilic blockcopolymers.

The term “metal salt” refers to a compound that includes a metal cationand one or more anions. Examples of metal salts include, for example,HAuCl₄, NaAuCl₄, Pd(NO₃)₂, PdSO₄, AgNO₃, H₂PtCl₆, or PdX₂ wherein X isCl, Br, or I, and the like.

As used herein, in connection with the surface of a micelle particle,the term “functional group” refers to the portion of a compound that canbe covalently bonded to the exterior of a micelle particle. Examples offunctional groups include cell targeting agents, water soluble polymers,such as PEG and its derivatives (e.g., as illustrated in FIG. 1, whereinn is about 10 to about 500), sugars, including modified sugars andsaccharides, fluorophores, and cell ligands such as biotin, folate, andthe like. The functional groups can be linked to the micelle particlesusing click chemistry, for example, by modifying the compound to includea —CH₂CH₂—N₃ group or —CH₂CH₂—SH group, at any suitable location.

The click reaction between alkynes and azides can be catalyzed by, forexample, various copper(I) catalysts. The copper(I) catalysts often areprepared from a copper(II) salt such as CuSO₄, CuCl₂, CuBr₂, or Cu(OAc)₂and a suitable reducing agent such as sodium ascorbate, copper metal, orhydrazine. Alternatively, copper(I) salts such as CuI, CuCl, or CuBr maybe used directly, in the presence or absence of another reducing agent.

Suitable photoinitiators for thiol-ene reactions include photoinitiatorswell known to those of skill in the art for thiol-ene reactions, such as2,2′-dimethoxy-2-phenylacetophenone, 1-hydroxy-cyclohexylphenylketone,benzophenone, 2-hydroxy-2-methyl-1-phenyl-1-propanone, and2-benzyl-2-(dimethylamino)-1-[4-(4-morpholinyl)phenyl]-1-butanone.

Development of Micelles and Formulations

Two widely used scaffolds in multivalency are dendrimers and goldnanoparticles protected with functionalized thiols. The methods ofproducing multivalent nanoparticles described herein are significantlyless expensive than the procedures for preparing dendrimers and goldparticles. The preparative methods described herein do not requireiterative synthesis (required for dendrimer preparation) and they do notrequire rare metals, such as gold. Additionally,surface-functionalization of the nanoparticles described herein can becarried out in “one-pot” after crosslinking, and stimuli-triggeredrelease mechanisms can be engineered into the nanoparticle system. Themethods are applicable to, for example, the preparation of micelles,organic or inorganic nanoparticles, liposomes, and/or globular orrod-like reversed micelles.

An ideal carrier system for anticancer drugs should have several of thefollowing features: (a) encapsulation of either water-soluble orwater-insoluble drugs, (b) suitable nanodimension (50-200 nm) formaximal exploitation of the EPR effect (for passive targeting), (c) asurface architecture that allows for facile multivalent surfacefunctionalization (e.g., with ligands or antibodies for activetargeting), (d) high encapsulating capacity, (e) good stability prior torelease, (f) fast release of encapsulated contents under appropriatestimulation (e.g., pH change), (g) simple preparation from readilyavailable starting materials, and (h) good biocompatibility and lowtoxicity. These stringent requirements make it extremely challenging todevelop effective delivery systems for anticancer drugs. Thecompositions and methods described can fulfill most, if not all, ofthese criteria, to create efficient drug delivery vehicles for bothwater-insoluble and water-soluble drugs, for example, anticancer drugs.

Micelles and Preparative Techniques

Radical polymerization of polymerizable surfactants (sufmers) is acommon method to prepare organic nanoparticles from micelles. Previousprocedures typically employ free radical polymerization of surfactantwith polymerizable groups in the head or tail. However, the resultingnanoparticles are exclusively linked by carbon-carbon bonds. Therefore,these nanoparticles lack many of the features, such as encapsulation,reversibility, controlled release, and surface functionalization, neededfor a suitable drug delivery system. Condensation polymerization may beused to crosslink a micelle, but this technique has significantdrawbacks. For example, it is difficult to obtain high yields in amideor ester condensation reactions at room temperature in water. Furthersurface functionalization is also difficult.

The methods described herein solve these problems by using speciallydesigned “clickable” surfactants taking advantage of highly efficientand water-compatible thiol-ene “click reaction”. They differ from theconventional crosslinking methods described above in several importantways, to provide novel crosslinked micelles.

The invention therefore provides delivery vehicles for eitherwater-soluble or water-insoluble drugs, controlled release devices,protective coatings for organic or inorganic nanoparticles,“nanoreactors” for templated synthesis of nanoparticles or nanowires,and/or a “housing” for catalytic metal nanoparticles.

The compositions and methods described herein also allow for targeteddrug delivery and controlled release of hydrophobic or hydrophilic cargomolecules, such as anticancer drugs. The size, surface-functionality,and release-mechanism of this new class of materials can be controlledby varying the methods, as described below.

The invention provides novel approach to crosslink surfactant micellesby a “covalent-capture” strategy. Surfactants, such as compound 1, canbe prepared in a few steps from simple, inexpensive starting materials(Scheme 1). The hydrophobic tail and the ammonium headgroup of compound1 allow its formation into micelles spontaneously in water above itscritical micelle concentration of 1.4×10⁻⁴ M. The three alkynyl groupson the ammonium allow the surface of the micelle to be readilycrosslinked with azido-containing crosslinkers (e.g., 2) by highlyefficient click reactions (FIG. 1).

Click chemistry (e.g., the 1,3-dipolar cycloaddition of an alkyne and anazide) is characterized by mild reaction conditions, nearly quantitativeconversion, and tolerance of a variety of functional groups (Scheme 2).

-   -   wherein R¹ is a portion of an amphiphilic surfactant and R² is a        portion of a crosslinker or surface modifying functional group,        or vice versa, or various groups as described herein.

Crosslinking of micelles is known, but previous procedures typicallyemploy free radical polymerization of surfactant with polymerizablegroups in the head or tail. The methods described herein differ fromthis conventional crosslinking in several important ways, to providenovel crosslinked micelles.

We found several advantages in using the thiol-ene click reaction inpreparing RMs.

The reaction is extremely efficient, even in highly demanding situationssuch as the synthesis of dendrimers and selective functionalization ofproteins.

Incorporation of vinyl (i.e., ene) groups is extremely simple in asurfactant. Cationic surfactant 10 (FIG. 2), for example, contains threevinyl groups in the headgroup and can be prepared easily fromcommercially available materials. Having three cross-linkable groups ina concentrated fashion enhances the cross-linking density at the coreand is advantageous to the stability of the cross-linked micelle.

There is great flexibility in the structure of thiol cross-linkers. Inparticular, if the distance between the thiol groups matches reasonablywell with the average distance between the surfactant headgroups in theRM, cross-linking should cause minimal disturbance to the packing of thesurfactants. Free radical polymerization of vinyl monomers, on the otherhand, tends to make the structure more compact.

The reaction has excellent tolerance for functional groups. A wide rangeof commercially available thiol cross-linkers may be used, andintroduction of additional functional groups to the RM isstraightforward.

The crosslinker may be tuned in length. The loss of order that oftenoccurs in radical polymerization can thus be avoided. After crosslinkingand termination (of any unreacted azide groups, e.g., by propargylalcohol 3, step (b) of FIG. 1), the Alkynyl-SCM obtained is about 8-10nm in diameter, as determined by dynamic light scattering. A variety ofother crosslinkers are illustrated in Schemes 6 and 11. The crosslinkersof Scheme 11 can be used, for example, when crosslinkable amphiphilessuch as the POPC mimic or the Lyso-PC mimic are used (Schemes 7-11).

Because a 1:1 stoichiometry can be used for 1 and 2 in the crosslinkingstep, the Alkynyl-SCM on average can contain approximately one unreactedalkynyl group per surfactant molecule after crosslinking. For a typicalmicelle that contains 50-100 surfactant molecules, the Alkynyl-SCM hastens of alkynyl groups for post-functionalization through further clickchemistry. For example, fluorophore 4, sugar 5, and water-solublepolymers 6 have been attached to the water-soluble organic nanoparticles(FIG. 1) to provide various surface-functionalized SCMs. Because clickchemistry was used in both crosslinking and post-functionalization, theprocedures can be carried out in “one-pot” at room temperature (˜23°C.). Although multifunctionalization is also possible with dendrimersand gold nanoparticles, the methods described herein are advantageousfor a number of reasons, including the spontaneous formation ofsurfactant micelles. The spontaneous micelle formation obviates the needfor stepwise synthesis, such as in dendrimer preparation. A furtheradvantage is the avoidance of expensive metals, such as rare metalcatalysts and gold nanoparticles, which translates to lower overallcosts for particle preparation, particularly on a large scale.

Reversible crosslinkers (e.g., 7 or 8, below) can be employed so thatthe SCMs can be destroyed after cleaving the disulfide or ketal bonds.For example, pyrene, a model hydrophobic guest, has been successfullytrapped inside the SCMs. Pyrene's different fluorescence in hydrophobicand hydrophilic environments allows for monitoring breakage of the SCMsby fluorescence. For SCMs prepared using reversible crosslinkers 7 or 8,experiments showed that entrapped pyrene was released completely within1 minute after the addition of HIO₄, orthiol 9, respectively. HIO₄ wasused to cleave the 1,2-diol in the crosslinker derived from 2, and thiol9 was used to cleave the disulfide bond in the crosslinker derived from7, to the corresponding SCMs. Crosslinking moieties derived fromacid-sensitive crosslinker 8 can be similarly cleaved in suitably acidicenvironments.

Crosslinking occurs exclusively at the micelle interface because of thealkynyl group locations. The interfacial crosslinking is applicable toother systems as well. For example, crosslinked reversed micelles (CRMs)have been prepared by “thiol-alkene” addition reactions, also known asthiol-ene click chemistry. See Scheme 3 and FIG. 2. Unlike the SCMs thatare soluble in water, the CRMs are soluble in nonpolar organic solventsbecause the hydrocarbon tails face outward.

The CRMs can also extract metal ions, such as aurate ions (e.g., AuCl₄⁻), from an aqueous solution into the organic phase as a result of themultiple ammonium headgroups in the interior. The entrapped aurate waseasily reduced by sodium borohydride to afford gold nanoparticles.

A distinct advantage of this approach is the ability to prepareextremely small metal nanoparticles (for example, if not all bromideanions are exchanged into aurate) and metal alloy nanoparticles (if twoor more metal precursors such as AuCl₄ ⁻ and PtCl₆ ²⁻ are used). Suchmetal nanoparticles are extremely difficult to prepare using othermethods. The metal nanoparticles have a variety of applications incatalysis and separation technology.

Encapsulation and Release of Camptothecin

Virtually any hydrophobic drug or reagent can be encapsulated within thecrosslinked micelles and nanoparticles described herein. Examples of twospecific drugs that can be encapsulated in the micelles include, forexample, camptothecin and tamoxifen.

Camptothecin (CAM) was isolated from a Chinese tree, Camptothecaacuminate, in 1966. Its high antitumor activity against a broad range ofexperimental tumors quickly made it a prominent lead in anticancer drugdevelopment. Because of its poor water-solubility, CAM entered clinicaltrials as the water-soluble sodium salt (CAM-Na). However, CAM-Na hasonly one-tenth of the biological activity of the parent drug. The severedose-dependent toxicities including vomiting, diarrhea, hemorrhagicenterocolitis, leucopenia, and thrombocytopenia, stopped the developmentof the original drug at phase-I trials. In the late 1980s, elucidationof the mechanism of CAM activity prompted renewed interest in this drug.

An effective carrier for CAM should enhance its water-solubility,protect it from hydrolysis into the inactive carboxylate form, have highselectivity for cancerous cells using both passive and active targeting,and release the drug under acidic conditions. The crosslinking methodsdescribed herein are especially suitable to meet these challenges.

CAM is a very hydrophobic molecule, having similar water insolubility topyrene. Because the interior of the SCMs is hydrophobic, CAM was readilysolubilized in water using SCMs. Both the hydrophobic environment andthe surface crosslinking protect the drug from hydrolysis. To takeadvantage of the EPR effect, the size of the drug carrier should ideallybe about 50-200 nm. Several SCRs were prepared as described herein andwere determined to have diameters of about 8-10 nm. Encapsulation ofcamptothecin can increase the size of the particle.

The reprecipitation method was first reported by Nakanishi andco-workers to prepare nanoparticles of water-insoluble organic compounds(Jpn. J. Appl. Phys. 1996, 35, L221-L223; J. Am. Chem. Soc. 2006,128(50), 15944-15945). In this method, a water-insoluble compound isfirst dissolved in a water miscible solvent, such as acetone, alcohol,or DMSO. The organic solution is then added to an aqueous solution via asyringe under rapid stirring. Large numbers of microcrystalline seeds(typically 20-30 nm in diameter) form immediately upon mixing. Within0.5-1 hours, these seeds typically aggregate and grow into largernanoparticles, often hundreds of nanometers in diameter. Reprecipitationhas been applied to many organic compounds, and has been scaled-upindustrially, as a suitable method to prepare nanosized organicparticles.

Preparation of CAM nanoparticles can be carried out in a similar manner,and as illustrated in FIG. 3. In the reprecipitation method, astabilizer is typically used to protect the surface of the organicnanoparticles from further growth. The stabilizer can be a water-solublepolymer or surfactant. Although surfactant 1 may be used for thispurpose, other surfactants, such as crosslinkable surfactant 11, with aphosphocholine-like headgroup, may also be used. An additional advantageis that phosphocholine lipids are the major components of mammalian cellmembranes, indicating that 11 is likely to be biocompatible. Thehydrophobic tail of 11 has high affinity for the nonpolar surface of CAMnanoparticles in water. Once the nanoparticles are coated withsurfactant 11, further particle size growth is prohibited and asignificant increase in colloidal stability is achieved.

The concentration of CAM in the organic solvent can be varied. DMSO isone suitable choice, although other solvents, such as acetonerethanol,may be also used. The rate of stirring, the incubation time, the amountof surfactant, temperature, and the timing of crosslinking (e.g., byaddition of the crosslinker and/or the copper catalyst), may also bevaried. These parameters influence the size and the crystallinity of theCAM nanoparticles, which affect their bioavailability andbiodistribution. The stability of the surface crosslinked nanoparticlescan be varied by altering the crosslinking density and can be controlledby the amount of crosslinker (e.g., 7 and 8) and by the reactionkinetics used in preparing the nanoparticles.

For biological applications, it may be desirable to employ a catalystother than copper ions. Copper power or wire can be suitably employed tocatalyze the click chemistry reactions and it can be easily removedafter reactions are complete. Alternatively, reprecipitation may becarried out at temperatures around 90° C., a temperature at which clickchemistry occurs readily without catalysts.

Poly(ethylene glycol), or “PEG”, can be clicked onto the surface of themicelles to avoid nonselective adsorption of proteins. PEGylation ofSCMs has been performed, for example, using azido PEG derivatives 6 (seeFIG. 1). The same procedure can be used to protect the surface of theCAM-SCMs.

At this stage of preparation, a single CAM particle has hundreds tothousands of drug molecules in the interior, and high loading efficiencyis therefore achieved. Surface-crosslinking with a water-solublesurfactant can ensure good solubility and stability. The dimensions ofthe nanoparticles (50-200 nm) allows their accumulation at the canceroussites based on the EPR effect. Surface PEGylation can ensure longcirculation time of the CAM-SCMs in the blood.

Folate receptors are overexpressed on the surface of cancerous cells.Active targeting can be achieved by functionalizing the micelle surfacewith targeting agents, such as folate, with the use of click chemistry.After crosslinking, azido-derivatives 12 and 13 can be clicked onto theCAM-SCM surface to afford the CAM-SCM Folate (FIG. 3). The 12/13 ratiocan be varied to adjust the surface-property of the nanoparticles. Theshort PEG derivative 13, or similar ligands, can be used instead of thepolymeric version 6 to make the folate ligands more accessible tocellular receptors.

Cancerous tissues are known to be more acidic than normal tissues.Acid-triggered release is therefore a suitable release mechanism fordelivery of encapsulated drugs. The acid-sensitive diazido compound 8can be used as an acid-sensitive crosslinker.

CAM is fluorescent, thus the release of CAM from the CAM-SCMs can bemonitored by fluorescence spectroscopy. Both aggregation andenvironmental polarity are known to affect the fluorescence of CAM. Thestability of the CAM-SCMs can be readily tuned by using a combination ofdifferent crosslinkers. Crosslinking moieties derived from crosslinker 2are completely stable under physiological conditions, whereas thedisulfide bond of a crosslinking moiety derived from crosslinker 7 canbe cleaved under reducing conditions provided to a cell. Mixing of 2, 7,and 8 allows for fine-tuning of the stability of the CAM-SCMs bothoutside and inside the cell.

Encapsulation and Release of Doxorubicin.

The crosslinking strategy may be used in liposomes, which canencapsulate hydrophilic drugs or agents. Examples of three specificdrugs that can be encapsulated in crosslinked liposomes includedoxorubicin (Dox), lurtotecan, and methotrexate.

Dox is currently used to treat a wide range of cancers, including acutelymphoblastic leukemia, acute myeloblastic leukemia, Wilms' tumor,neuroblastoma, soft tissue and bone sarcomas, breast carcinoma, ovariancarcinoma, transitional cell bladder carcinoma, thyroid carcinoma,gastric carcinoma Hodgkin's disease, malignant lymphoma, andbronchogenic carcinoma. Unlike CAM, Dox is completely water-soluble. TheFDA's information website for Dox states that the initial distributionhalf-life of approximately 5 minutes suggests rapid tissue uptake . . .while its slow elimination from tissues is reflected by a terminalhalf-life of 20 to 48 hours. Bioavailability is therefore not a concern,however improved methods of active targeting and controlled release areneeded for Dox delivery.

A Dox delivery strategy was developed using the crosslinking methodsdescribed herein, combined with liposomal delivery. One benefit of thisapproach is the facile integration of the micelle technology withcommercial products. Dox liposomes are currently used commercially. TheDox liposomes are well suited for targeted drug-delivery because theliposome membranes are made of the same lipids of biomembranes andsurfaces decorated with receptor-specific ligands. PEGylation of the doxliposome surfaces increases the circulation time and reduces proteinadsorption. However, premature leakage is a problem for the Doxliposomes because of the non-covalent liposome structure. Additionally,controlling the release of liposomal cargo is difficult.

The crosslinking methods described herein address both of thesechallenges concurrently. Instead of single-tailed surfactant,alkyne-functionalized phosphocholine derivative 14 can be prepared usingstandard synthetic techniques. Lipid 14 may be used with the commonphospholipid POPC (palmitoyl-oleoyl phosphatidylcholine, 15) to formnegatively charged liposomes.

Carboxyfluorescein (CF) is a water-soluble dye. To investigatecontrolled release of liposomal content, CF was used as a model drug forhydrophilic chemotherapeutic agent. CF-leakage assays are well known andare widely used in liposomal chemistry. In this assay, largeunimolecular vesicles (LUVs) are first prepared in the presence of aself-quenching concentration (>50 mM) of CF. The external, untrapped CFcan then be removed by gel permeation chromatography. If CF leaks out ofthe LUVs, it becomes diluted and then fluoresces more intensely.

Controlled release of CF from the LUVs is illustrated in FIG. 20.CF-containing LUVs can be prepared with either 14, or a mixture of 14with other non-crosslinkable lipids (e.g., POPC/POPG). Click reactionswith redox-sensitive 7 can crosslink mainly the outer leaflet of thelipid bilayer. When the surface crosslinked LUV (SCL-LUV) is placed in aconcentrated salt solution (e.g., 5 M NaCl), the liposomes experienceosmotic stress. With a sufficiently high crosslinking density, theSCL-LUV can withstand the osmotic pressure and keep CF inside. Uponcleavage of the crosslinker under reducing conditions, the noncovalentlylinked LUV ruptures easily, releasing the entrapped contents. The samemethod may be used with acid-sensitive 8 as the crosslinker, but the CFassay is not suitable under acid conditions. Other assays such asANTS/DPX assay may be more suitable to monitor the controlled release.

The relationship between liposomal stability and crosslinking densitycan be studied systematically by varying the 14/15 in the lipidformation. Highest crosslinking, and thus highest stability, can beachieved for the SCL-LUV with 14 as the only lipid, whereas nocrosslinking is achieved if only 15 is employed. Stability of theSCL-LUV can be measured by either the maximum osmotic pressure it cantolerate without leakage, or by the percent leakage of CF over timeunder a given osmotic pressure.

The basic strategies for the passive and active targeting are the samein the CAM-SCMs (FIG. 3). Briefly, passive targeting is achieved bycontrolling the size of the liposomes (100-200 nm) and PEGylation (toavoid protein adsorption), and active targeting bypost-functionalization by folate derivative 12. These procedures providethe ability to control the size, stability, surface-functionalization ofthe particles, and the release of both water-insoluble and water-solubleanticancer drugs from the particles.

Micelle Embodiments

The invention therefore provides a variety of novel organic particle. Insome embodiments, the particle can have surface crosslinkednon-polymeric organic amphiphiles. Polar head groups of the amphiphilescan be covalently crosslinked to each other at the surface of theparticle through triazole groups or thioethers groups. The tail groupsof the amphiphiles can then be arranged toward the interior of theparticle. These particles can be water-soluble.

Amphiphiles used to prepare the particles. In some embodiments, theparticles are spheroid and comprise about 10 to about 150 crosslinkedamphiphiles and about 10 to about 150 crosslinking moieties. In otherembodiments, the particle can include larger amounts of amphiphiles andcrosslinking moieties, such as 20 to about 200, to about 250, to about500, or to about 1000. When the particles are prepared as rod-likeshaped particles, the particles are typically prepared from a largernumber of amphiphiles, such as about 100 to about 1000, or about 500 toabout 5000 amphiphiles. The ratio of amphiphiles to crosslinkingmoieties can be from about 1:10 to about 10:1, or any integer ratio inbetween those ratios.

The tail groups of the amphiphiles can be non-polar groups such as alkylgroups, fluoroalkyl groups, or a combination thereof. The alkyl orfluoroalkyl groups can contain any suitable number of carbons such thatthe amphiphiles self-assemble and form micelles. Accordingly, in someembodiments the tail group can include about 6 to about 50 carbon atomsin a chain, wherein the chain can be straight or branched. The groupscan also be (C₂₀-C₅₀) alkyl groups or fluoroalkyl groups, (C₆-C₂₅) alkylgroups or fluoroalkyl groups, (C₆-C₂₂) alkyl groups or fluoroalkylgroups, (C₆-C₂₀) alkyl groups or fluoroalkyl groups, or (C₈-C₁₈) alkylgroups or fluoroalkyl groups. The tail groups can optionally include agroup that links the tail to the head group of the amphiphile or thatlinks the tail group to another section of the tail group. The linkinggroup can be, for example, an ester, imine, boronate, or disulfidegroup. The linking group can also be a salt bridge, such as aguanidinium-carboxylate or guanidinium-phosphate salt bridge.

In some embodiments, the tail groups are non-polar alkyl or fluoroalkylchains on the interior of the particle and the head groups are polargroups at the surface of the particle. In such embodiments, the particlehas a hydrophobic core and a hydrophilic exterior. A non-polar tail maybe removed by hydrolysis if it is connected to the head group by ester,imine, boronate, or other hydrolyzable linkage, to provide a polar tail.The tail may be partly or completely destroyed if it consists offunctional groups that can be degraded. For example, an unsaturatedhydrocarbon tail may be degraded by oxidation, or the tail may beremoved by reduction if it is connected to the head group by disulfidebonds. The tail may also be removed if it is connected to the head groupby noncovalent bonds such as guanidinium-carboxylate orguanidinium-phosphate salt bridges. Thus, in other embodiments, such aswhen interior tail groups are non-polar and a hydrolyzable linking groupis hydrolyzed to provide a polar tail moiety, the tail groups of theamphiphiles are polar and the particle has a hydrophilic core and ahydrophilic exterior. Accordingly, the tail groups of the amphiphilesarranged toward the interior of the particle can be non-polarhydrocarbon or fluorocarbon tail groups, polar tail groups, or acombination thereof.

Properties and forms of the Organic Particles. The organic particle canbe in the form of spheres or rods. The particles can also be in the formof a liposome or vesicle having a bilayer of amphiphiles, where thebilayer includes one or more water compartments between the bilayer ofamphiphiles. The preparation of vesicles is described in Example 5below.

Particle Cargo and Appendages. The organic particle can include one ormore cargo molecules within the particle or at the surface of theparticle. The cargo can be free within the interior of the particle orcovalently bonded to a tail group of an amphiphile. The cargo can alsobe bonded to the surface of the particle though electrostaticinteractions, or the cargo molecules can be optionally covalently bondedto the surface of the particle, for example, through a linking groupsuch as a group that has been bonded to a surface functional group(e.g., an alkyne, azide, alkene, or thiol moiety) through a clickreaction.

The cargo molecules can be a drug, an organic nanoparticle, an inorganicnanoparticle, a fluorophore, a diagnostic agent, and/or a catalyst. Thecatalyst can be an organic molecule catalyst, an organometalliccatalyst, or a transition metal catalyst. In some embodiments, thecatalysts are phosphine-stabilized rhodium catalysts for catalytichydrogenation and/or hydroformylation. In some embodiments, thecatalysts are phosphine-stabilized palladium catalysts formetal-catalyzed cross-coupling. In other embodiments, the catalysts aremetallosalen complexes for catalytic epoxidation or other reactions.

The surface of the particle can include one or more surface groups suchas alkynes, alkenes, azides, aldehydes, or alcohols, or attached groupssuch as water-soluble polymers, fluorophores, biological ligands,nucleic acids, nucleic acid analogues, catalysts, or a combinationthereof. The biological ligands can be active targeting agents, sugar orpeptide moieties, or analogues thereof. The surface groups can bereceptors for ligands on a biological host, or ligands for receptors ona biological host. The biological host can be a bacterium, a virus, or aeukaryotic cell. The surface functional groups can be attached to thesurface of the particle by using click reactions with appropriatelyfunctionalized groups above. The surface of the particle can befunctionalized with about 10 to about 150 functionalized surface groups;or about 100 to about 5000 functionalized surface groups.

The surface crosslinking can be reversible or degradable. For example,in some embodiments the surface crosslinking can be cleaved by heat, bya change in pH, by a reducing agent, or by a combination thereof.Accordingly, the cleavage can be under chemical conditions includingcontacting the particle with a reducing agent, an acid, or a periodatecompound, and the like.

Crosslinked Reverse Micelle Particles. In some embodiments, the organicparticles can be considered crosslinked reverse micelle particles. Theamphiphiles used to prepare these particles can be the same as used forthe surface crosslinked micelle particles described above, however theparticles are prepared differently, thereby providing different physicaland chemical properties to the particles. Thus, the invention providesan organic particle comprising non-polymeric crosslinked amphiphiles;where the amphiphiles comprise one or more nonpolar alkyl or fluoroalkylchains and one or more polar head groups; the nonpolar chains arelocated on the exterior of the particle and the polar head groups areoriented toward the interior of the particle; and the amphiphiles arecovalently crosslinked to each other near the head groups throughtriazole groups or thioether groups. The particles can be a crosslinkedreverse micelle (CRM) particle and the particle can be soluble inorganic solvents.

The organic particle can contain one or more metal salts or metalparticles within the particle. The particle can have one or morecatalytically active groups oriented toward the interior of theparticle, such as on a polar head group. The catalytically active groupscan be one or more of carboxylic acids, sulfonic acids, amines, orthiols, where the catalytically active groups are covalently bonded toone or more of the amphiphiles.

Surface Crosslinked Organic Particles Prepared by Azide-Alkyne ClickReactions. The invention provides methods for preparing surfacecrosslinked organic particles, as described in the summary above, byinducing cycloaddition between the alkynes and azides, thermally or witha suitable catalyst, to covalently crosslink the amphiphiles to eachother near the head groups through formation of triazole groups. Theamphiphiles can self-assemble through hydrophobic interactions among thehydrophobic groups. In some embodiments, the amphiphiles include one ormore non-polar alkyl tails and a tripropargylammonium head group or adipropargyl(alkyl)ammonium head group. The surface-crosslinked particlecan be water soluble. The catalyst used to facilitate the click reactioncan be a copper catalyst, such as a Cu(I) salt or a Cu(II) salt reducedin-situ to Cu(I).

In some embodiments, the crosslinking agents comprise two or more azidogroups when the polar head groups comprise alkynyl groups, or two ormore alkynyl groups when the polar head groups comprise azido groups.When preparing the particles, the amphiphiles and water can be in thepresence of one or more cargo molecules. The cargo molecules are therebyencapsulated in the hydrophobic core upon formation of theself-assembled structure. In some embodiments, the cargo molecules arehydrophobic. In other embodiments, hydrophobic tails of the amphiphilesat the interior of the particle are hydrolyzed from esters to carboxylicacid or hydroxyl groups, and the cargo molecules are hydrophilic. Insome embodiments, the amphiphiles and water are in the presence of oneor more cargo molecules, the particles are in the form of vesicles, andthe cargo molecules are encapsulated in water compartments of thevesicles.

In preparing the particles, the methods can further include contactingthe surface-crosslinked particle with one or more azido-containing oralkynyl-containing compounds that are water-soluble polymers,fluorophores, biological ligands, nucleic acids or analogues thereof, ora combination thereof. Cycloaddition can be induced between alkynes orazides on the surface of the particle with the azido-containing oralkynyl-containing compounds. The cycloaddition can be induced thermallyor with a suitable catalyst to provide a water soluble multivalentparticle that has a plurality of water-soluble polymers, fluorophores,biological ligands, nucleic acids or analogues thereof, or a combinationthereof, linked to the surface of the particle through triazole groups.

Surface Crosslinked Organic Particles Prepared by Thiol-ene ClickReactions. The invention provides methods for preparingsurface-crosslinked organic particles, as described in the summaryabove, by inducing thiol-ene addition between alkenes of the amphiphilesand thiol groups of the crosslinkers photochemically to covalentlycrosslink the amphiphiles to each other near the head groups through theformation of thioether groups. In some embodiments, the amphiphilesinclude one or more non-polar alkyl tails and a triallylammonium headgroup, to provide a self-assembled water soluble micelle. The inductionof the thiol-ene addition between the alkenes of the amphiphiles and thethiol groups can be carried out in the presence of a photoinitiator.

The particles can be prepared such that the amphiphiles and water are inthe presence of one or more cargo molecules. The cargo molecules arethereby encapsulated in the hydrophobic core upon formation of theself-assembled structure. In some embodiments, the cargo molecules arehydrophobic, and in other embodiments, the cargo molecules arehydrophilic. In other embodiments, the amphiphiles and water are in thepresence of one or more cargo molecules, the particles are in the formof vesicles, and the cargo molecules are encapsulated in watercompartments formed within the vesicles. As discussed above, the cargomolecules can be one or more of drugs, organic nanoparticles, inorganicnanoparticles, fluorophores, diagnostic agents, and catalysts.

In some embodiments, the method further includes contacting thesurface-crosslinked particle with one or more thiol-containing compoundscomprising water-soluble polymers, fluorophores, biological ligands,nucleic acids or analogues, or a combination thereof. Thiol-ene additionreactions can be induced between the thiol groups of thethiol-containing compounds and alkene groups at the surface of thesurface-crosslinked particle to provide a water soluble multivalentparticle that has a plurality of surface functional group compoundslinked to the surface of the particle through thioether groups. Thesesurface groups can be water-soluble polymers, fluorophores, biologicalligands, nucleic acids or analogues thereof, or a combination thereof,linked to the surface of the particle through thioether groups. Thebiological ligands can include sugar or peptide moieties, or analoguesthereof

Additional Methods of the Invention. The invention also provides methodsto form metal nanoparticles. A metal salt and a plurality of particlesdescribed above, such as a particle with a hydrophilic interior, can becontacted in an aqueous/organic solvent mixture, thereby extractingmetal ions of the metal salt into the organic solvent. The metal ionsthen migrate to the interior of the particle, to provide a crosslinkedorganic particle encapsulating metal ions. The crosslinked organicparticle encapsulating metal ions can be contacted with a reducingagent, thereby reducing the metal ions in the interior of thecrosslinked organic particle, to provide the metal nanoparticles. Themetal salt can include metals such as AuX₄ ⁻, PtX₆ ²⁻, PdX₄ ²⁻, where Xis a halogen, for example, AuCl₄ ⁻, PtCl₆ ²⁻, PdCl₄ ²⁻, or a combinationthereof. More than one type of metal salt can contacted with thecrosslinked organic particle in order to form alloys. In someembodiments, the metal nanoparticle formed has a diameter of 1 nm (orabout 3-4 atoms) to about 100 nm, or about 1.5 nm to about 10 nm, orabout 2 nm to about 5 nm.

The invention further provides a therapeutic method comprisingadministering to a patient in need therapy an effective amount of thedelivery system that includes a plurality of particles described above,where the surface crosslinking of the particles encapsulate one or moredrugs, the surface crosslinking of the particles is cleaved in vivo, andthe drug of the particles is released into the body of the patient,thereby providing the drug to the patient. In some embodiments, acrosslinked micelle is used for the delivery of hydrophobic drugs. Inother embodiments, a crosslinked vesicle is used for the delivery ofhydrophilic drugs.

While various embodiments of the invention have been described above,the invention also provides the following embodiments. In oneembodiment, the invention provides a multivalent surface-crosslinkedmicelle (SCM) particle comprising about 10 to about 150 crosslinkedamphiphiles and about 10 to about 150 crosslinking moieties. Thecrosslinkiner moieties, such as those derived from the compoundsillustrated in Schemes 6 and 11 below, can link one amphiphile toanother, thereby stabilizing the micelle by the formation of triazolelinkages, or thioether linkages, resulting from the click chemistryreactions used to crosslink the amphiphiles.

The amphiphiles include non-polar alkyl chains, such as (C₁₀-C₂₀)alkylchains, on the interior of the particle and polar head groups at thesurface of the particle. The head groups can be tripropargylammoniumgroups or tryallylammonium groups that are covalently crosslinked toeach other by the crosslinkers, thereby forming triazole groups orthioether groups. The head groups orient themselves at the surface ofthe particle, thereby making the particle water soluble.

The particle can have one or more drugs or diagnostic agentsencapsulated within the particle. Examples of such drugs includecamptothecin and doxorubicin. Accordingly, the encapsulated agents,i.e., the drug or diagnostic agent, can be hydrophobic or hydrophilic.

The surface of the particle can have functionalized with about 10 toabout 150 alkyne or alkene groups available for linking to surfacemodifying groups that include various functional groups. The functionalgroups can be water-soluble polymers, sugars, fluorophores, activetargeting agents, or a combination thereof. The functional groups can bebinding sites for a bacteria, virus, or tumor cell.

Any of the crosslinked micelles described herein may be crosslinked withreversible crosslinkers. For example, when the surface of the micellecrosslinked with reversible crosslinkers is contacted with anappropriate acid, reducing agent, or diol cleaving agent, thecrosslinkers are cleaved, thereby eliminating the crosslinking andallowing encapsulated agents to be released from the micelle. Thus theparticles can be used for the controlled release of drugs, such ascamptothecin or doxorubicin, among other agents. Accordingly, theinvention provides a deliver system that includes a plurality ofmultivalent particles described herein, and a pharmaceuticallyacceptable diluent or carrier.

The invention also provides a crosslinked reverse micelle (CRM) particlethat includes about 10 to about 150 crosslinked amphiphiles and about 10to about 150 crosslinking moieties. The amphiphiles of the micelle havenon-polar alkyl chains, such as (C₁₀-C₂₀)alkyl chains, facing theexterior of the particle and polar head groups at the interior of theparticle. The head groups at the interior of the particle are covalentlycrosslinked to each other by crosslinkers through triazole groups orthioether groups, and the particle is soluble in organic solvents, suchas alkane solvents, acetone, DMSO, and the like.

Crosslinked reverse micelle particle can include one or more metal saltsor metal particles within the particle. Examples of such particlesinclude gold nanoparticles, palladium nanoparticles, or alloys thereof.

The invention also provides a method for preparing a surface-crosslinkedmicelle (SCM) particle. The method can include combining a plurality ofamphiphilic organic compounds and water, wherein the amphiphilic organiccompounds comprise one or more non-polar alkyl tails and atripropargylammonium head group, to provide water soluble micelles. Themicelles can be contacted with a suitable copper catalyst and adiazido-functionalized crosslinking agent, to provide thesurface-crosslinked micelle particle. The contacting, which results in aclick reaction, can be aided by the addition of a salt, such as sodiumascorbate. The resulting particle typically includes about 10 to about150 crosslinked amphiphiles and about 10 to about 150 crosslinkingmoieties. The non-polar alkyl chains are oriented to the interior of theparticle and polar head groups are at the surface of the particle. Thehead groups at the surface of the particle are covalently crosslinked toeach other by crosslinkers through triazole groups, and thesurface-crosslinked micelle has about 10 to about 150 alkyne groups atthe surface of the particle.

The amphiphilic organic compounds can form micelles in the presence of ahydrophobic drug or diagnostic agent. The resulting particles cantherefore include a hydrophobic drug or diagnostic agent encapsulated bythe surface-crosslinked micelle.

The method can also include functionalizing the surface of the particleby contacting the surface-crosslinked micelle particle with a suitablecopper catalyst and one or more azido-containing functional groupcompounds to form linkages to the functional groups using clickchemistry. The functional groups can include, for example, water-solublepolymers, sugars, fluorophores, active targeting agents, or acombination thereof. The resulting particles are water solublemultivalent surface-crosslinked micelle particles that have a pluralityof functional group compounds linked to the surface of thesurface-crosslinked micelle particle through triazole groups.

In another embodiment, the invention provides a method for preparing asurface-crosslinked micelle (SCM) particle by combining a plurality ofamphiphilic organic compounds and water, wherein the amphiphilic organiccompounds comprise one or more non-polar alkyl tails and atriallylammonium head group, to provide water soluble micelles. Themicelles can then be irradiating the micelles in the presence of aphotoinitiator and a dithio-functionalized crosslinking agent, toprovide surface-crosslinked micelle particles. The surface-crosslinkedmicelle particle typically include about 10 to about 150 crosslinkedamphiphiles and about 10 to about 150 crosslinking moieties. Thenon-polar alkyl chains are oriented to the interior of the particle andpolar head groups are at the surface of the particle, the head groups atthe surface of the particle are covalently crosslinked to each other bycrosslinkers through thioether groups, and the surface-crosslinkedmicelle particle has about 10 to about 150 alkene groups at the surfaceof the particle.

The amphiphilic organic compounds can form micelles the presence of ahydrophobic drug or diagnostic agent. The hydrophobic drug or diagnosticagent is thereby encapsulated by the surface-crosslinked micelle.

The method can further include irradiating the surface-crosslinkedmicelle particle in the presence of a photoinitiator and one or morethiol-containing functional group compounds to modify the surface of theparticle. The functional groups can include water-soluble polymers,sugars, fluorophores, active targeting agents, or a combination thereof.The resulting particle is a water soluble multivalentsurface-crosslinked micelle particle that has a plurality of functionalgroup compounds linked to the surface of the surface-crosslinked micelleparticle through thioether groups.

In yet another embodiment, the invention provides a method for preparinga crosslinked reverse micelle (CRM) particle. The method can includecombining a plurality of amphiphilic organic compounds, water, and asuitable organic solvent mixture. The organic solvent mixture caninclude a halogenated organic solvent, such as methylene chloride and/orchloroform, and a (C₆-C₁₂)alkane solvent, such as hexane, heptane, oroctane. The amphiphilic organic compounds can include one or morenon-polar alkyl tails and a triallylammonium head group, therebyproviding the reverse micelles; wherein the reverse micelles compriseamphiphiles that have non-polar alkyl chains on the exterior of theparticle and polar head groups at the interior of the particle. Theparticles can be irradiated in the presence of a photoinitiator and adithio-functionalized crosslinking agent, to provide the crosslinkedreverse micelle particles. The particle typically includes about 10 toabout 150 crosslinked amphiphiles, about 10 to about 150 crosslinkingmoieties linking the amphiphiles together. The non-polar (C₁₀-C₂₀)alkylchains are oriented to the exterior of the particle and polar headgroups are at the surface of the particle, the head groups at thesurface of the particle are covalently crosslinked to each other bycrosslinkers through thioether groups, and the particle is soluble inorganic solvents.

The crosslinking agent used in any of the methods above can bereversible crosslinking agent, which can be cleaved in the presence of,for example, a reducing agent, an acid, or a periodate compound.

In another embodiment, the invention provides a method of forming ametal nanoparticle. The method can include contacting a metal salt and aplurality of reversed micelle particles, in an aqueous/organic solventmixture, thereby extracting the metal cation of the metal salt into theorganic solvent, to provide crosslinked reverse micelles encapsulatingmetal cations. After separation from the aqueous solvent, thecrosslinked reverse micelles encapsulating metal cations can becontacted with a reducing agent, thereby reducing the metal cation inthe interior of the crosslinked reverse micelles, to provide the metalnanoparticle. The metal salt can include, for example, AuCl₄ ⁻, PdCl₄²⁻, or a combination thereof. More than one type of metal salt can bepresent in the aqueous/organic solvent mixture that is contacted withthe crosslinked reverse micelles, resulting in the formation of a metalnanoparticle that is an alloy of two or more different metals.

The invention further provides a therapeutic method that includesadministering to a patient in need therapy an effective amount of drugor diagnostic agent encapsulating particles described herein. Theparticles can be a deliver system as described above, wherein thesurface crosslinking of the micelles encapsulate one or more drugs, thesurface crosslinking of the micelles is reversible, the surfacecrosslinking is reversed in vivo, and the drug of the micelle isreleased into the body of the patient, thereby providing therapy to thepatient.

In some embodiments, the amphiphiles used to form the micelles describedherein can be a compound of formula I:

wherein T is a hydrophobic (C₁₀-C₂₀)alkyl tail connected to L through acovalent linkage such as an ether, ester, amine, amide, siloxane, imine,carbamate, urea, disulfide, thiother, or a direct bond:

L is a linking group, wherein the linking group comprises-Ph-, >N—C(O)—, —O—C(O)—, one to six methylene groups, or a combinationthereof, or L is a direct bond;

n is one, two, or three;

the dashed lines are optional bonds, which form alkynes when present oralkenes when absent; and

X is a suitable counter ion, such as halo, for example, F, Cl, Br, or I.

Examples of suitable crosslinker compounds of Formula I are illustratedin Scheme 5 below.

Crosslinkers can be crosslinked by click reactions using crosslinkingagents, such as those illustrated in Scheme 6.

Other crosslinked micelles can be prepared from phospholipid-containingamphiphiles, such as those illustrated below in Schemes 7-10.

In some embodiments, the amphiphiles can have two or three azido groupsand the crosslinkers can include two or three alkyne groups, such asthose illustrated in Scheme 11.

In some embodiments, the micelles can be formed in the presence of aswelling agent, to increase the size of the micelles. One example of asuitable swelling agent includes mesitylene. Examples of additionalamphiphiles that can be crosslinked are illustrated in Schemes 12-14below.

Anti-Cancer Formulations

The micelles described herein can be used to encapsulate anticancerdrugs, such as camptothecin and doxorubicin. The micelles provide for acontrolled release of the drugs under mild acidic conditions.Acid-triggered release is ideal for anticancer drugs because canceroustissue is more acidic than normal tissue. The size of the drug carriercan be tuned to maximize the accumulation of the drugs at canceroussites. Active targeting ligands, such as folate groups, can be attachedto the surface of the drug carriers to provide improved therapeuticeffectiveness.

Pharmaceutical Formulations

The crosslinked micelles or liposomes described herein, for example,those that include an encapsulated drug or diagnostic agent, can be usedto prepare therapeutic pharmaceutical compositions. The crosslinkedmicelles or liposomes can be formulated as pharmaceutical compositionsand administered to a mammalian host, such as a human patient, in avariety of forms. The forms can be specifically adapted to a chosenroute of administration, e.g., oral or parenteral administration, byintravenous, intramuscular, topical or subcutaneous routes.

The crosslinked micelles or liposomes may be systemically administeredin combination with a pharmaceutically acceptable vehicle, such as aninert diluent or an assimilable edible carrier. For oral administration,the crosslinked micelle or liposome compositions can be enclosed in hardor soft shell gelatin capsules, compressed into tablets, or incorporateddirectly into the food of a patient's diet. The crosslinked micelles orliposomes may also be combined with one or more excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, and the like. Such compositionsand preparations typically contain at least 0.1% of active agent. Thepercentage of the compositions and preparations can vary and mayconveniently be from about 2% to about 60% of the weight of a given unitdosage form. The amount of active agent in such therapeutically usefulcompositions is such that an effective dosage level can be obtained.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: binders such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; and a lubricant such as magnesium stearate. A sweeteningagent such as sucrose, fructose, lactose, or aspartame; or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring, maybe added. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose, or fructose as asweetening agent, methyl and propyl parabens as preservatives, a dye,and flavoring such as cherry or orange flavor. Any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations anddevices.

Drug encapsulating micelles or liposomes may be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the micelles or liposomes can be prepared in water, optionally mixedwith a nontoxic surfactant. Dispersions can be prepared in glycerol,liquid polyethylene glycols, triacetin, or mixtures thereof, or in apharmaceutically acceptable oil. Under ordinary conditions of storageand use, preparations may contain a preservative to prevent the growthof microorganisms.

Pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions, dispersions, or sterile powderscomprising the active ingredient adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. The ultimate dosage form should besterile, fluid, and stable under the conditions of manufacture andstorage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions, or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thiomersal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by agents delayingabsorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating themicelles in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, methods of preparation can includevacuum drying and freeze drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

Useful dosages of the compounds described herein can be determined bycomparing their in vitro activity, and in vive activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949 (Borch et al.). The amount of micelles required foruse in treatment will vary not only with the particular drugencapsulated but also with the route of administration, the nature ofthe condition being treated, and the age and condition of the patient,and will be ultimately at the discretion of an attendant physician orclinician.

The micelles or liposomes can be conveniently administered in a unitdosage form, for example, containing 5 to 1000 mg/m², about 10 to 750mg/m², or about 50 to 500 mg/m² of micelle per unit dosage form. Thedesired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four, or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

The invention thus provides therapeutic methods of treating cancer in amammal, which involve administering to a mammal having cancer aneffective amount of a micelle composition described herein. Mammalsinclude primates, humans, rodents, canines, felines, bovines, ovines,equines, swine, caprines, and the like. Cancer refers to any varioustype of malignant neoplasm, for example, colon cancer, breast cancer,melanoma and leukemia, or other cancerous conditions recited herein, andin general is characterized by an undesirable cellular proliferation,e.g., unregulated growth, lack of differentiation, local tissueinvasion, and metastasis.

The ability of a micelle or liposomes described herein to treat cancermay be determined by using assays well known to the art. For example,the design of treatment protocols, toxicity evaluation, data analysis,quantification of tumor cell kill, and the biological significance ofthe use of transplantable tumor screens are known.

The following Examples are intended to illustrate the above inventionand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the invention could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the invention.

EXAMPLES

Surface-crosslinked micelles were prepared as described herein.Post-functionalization, encapsulation, and controlled release studieswere carried out, as further described below.

Example 1 Facile Synthesis of Multivalent Water-Soluble OrganicNanoparticles Via “Surface-Clicking” of Alkynylated Surfactant Micelles

Crosslinking of alkyne-functionalized surfactants by azido crosslinkersthrough the highly efficient click reaction afforded surface-crosslinkedmicelles (SCMs). Post-functionalization of these water-solublenanoparticles was conveniently catalyzed by the residual Cu(I) catalystin the solution after addition of azide-functionalized polymers orligands (FIG. 4). The simplicity of the method, the extremely easysynthesis of the starting materials, and the utility of click reaction,make these materials an excellent platform for synthetic multivalentligands.

Multivalent interactions occur frequently between biological entities.When strong binding is not possible with a single receptor-ligand pair,multivalency, or simultaneous binding between multiple receptors andligands, becomes an effective strategy to enhance the binding.Significant efforts have been devoted in recent years to syntheticmultivalent ligands and their interactions with biological hosts. Two ofthe most widely used scaffolds in multivalency are dendrimers and goldnanoparticles protected with functionalized thiols.

An extremely simple method to prepare water-soluble organicnanoparticles as a new platform for multivalent ligands is describedherein. The highly efficient “click reaction” (Rostovtsev, Green; Fokin;and Sharpless; Angew. Chem., Int. Ed. 2002, 41, 2596-2599; Tomoe et al.,J. Org. Chem. 2002, 67, 3057-3064) was used to crosslink the surface ofalkynylated surfactant micelles. The resulting nanoparticles arecompletely water-soluble and contain numerous alkynyl groups on thesurface, and can be readily decorated with desired ligands. Althoughcrosslinking of surfactant micelles has been reported as early as in the1970s, the commonly utilized free radical polymerization offers no easyway to functionalize the resulting nanoparticles.

Cationic surfactant 1 was prepared in a few simple steps fromcommercially available 4-hydroxybenzaldehyde, 1-bromododecane, andtripropargyl amine. With an ammonium head group and a long hydrocarbontail, it forms micelles above 1.5×10⁻⁴ M in water. The design of thesurfactant puts numerous alkynyl groups on the surface of the micelles,which are readily crosslinked by azido derivatives, such as 2a-2c, inthe presence of Cu(I) catalyst (FIG. 21). The combination of CuSO₄ andsodium ascorbate quickly produced precipitates from a 10 mM micellarsolution of 1. Replacement of the CuSO₄ with CuCl₂, on the other hand,afforded a nearly transparent solution.

Choice of the crosslinker has a significant effect on the resultingmicelles. Water-soluble crosslinker 2a readily affordedsurface-crosslinked micelles (SCMs) 8-10 nm in diameter, according todynamic light scattering (DLS). Even though DLS also confirmed theformation of SCMs with 2b, much of this water-insoluble crosslinkerremained unconsumed even after prolonged reaction time, presumablybecause the two reactants were located in different phases. The reactionwas typically performed with a 1:1 mixture of 1 and 2a (typically at1-10 mM in water) with 2.5 mol % of CuCl₂ and 25 mol % of sodiumascorbate at room temperature (˜23° C.) for 24 hours. Propargyl alcoholwas added at the end to terminate the click reaction by consumingresidual azido groups left on the SCMs.

In addition to DLS, formation of SCMs can be monitored by ¹H NMRspectroscopy. The proton signals for a 10 mM solution of 1 in D₂O (FIG.5a ) were slightly broader than in CDCl₃. The peak broadening is anindication for micellization. Addition of 2a caused no change to thesignals of 1 (FIG. 5b ), but, after 24 hours in the presence of a Cu(I)catalyst, afforded extremely broad methyl and methylene peaks (δ=1.0-1.3ppm) of the dodecyl chain and nearly completely suppressed the signalsfrom the protons near the ammonium head group (FIG. 5c ). These effectswere not caused by the paramagnetic copper, because extensive dialysisof the sample against water removed the impurities and clarified thespectrum near 8=3-4 ppm, however the spectrum looked comparable (FIG. 5d).

Crosslinking thus brought significant changes to the intensity ofdifferent protons in the surfactant. In comparison to the proton signalsprior to crosslinking (FIG. 5a or 5 b), the loss of signal intensityfollowed the order of terminal CH₃<dodecyl CH₂<aromatic ArH<benzylicCH₂. In other words, the farther the proton is from the crosslinkingsite, the more its NMR signal was preserved. This result is expectedbecause the ends of the dodecyl chains maintain a fair level of mobilityinside the SCM, making them more visible in NMR spectroscopy than thosethat are restricted by the crosslinking. Note that the aromatic peaks ofthe SCM shifted upfield slightly, as a result of the proximity of thearomatic groups after crosslinking.

Additional insights on the crosslinking were obtained by cleaving thegeminal diol group in the crosslinker. After treatment with an excess ofperiodic acid, the alkylnyl-SCMs were subjected to ESI-MS analysis.Although many species were detected, the base peak was ammonium cation3, in line with the 1:1 stoichiometry between 1 and 2a used in thereaction. A smaller amount of 4 was also found, suggesting that, in someof the surfactants, all three triple bonds underwent the cycloaddition.

The presence of multiple alkynyl groups on the surface of the SCMs makesit easy to functionalize these nanoparticles. After crosslinking, anazido PEG derivative (5, m.w.=2000) was added directly to thealkynyl-SCM solution. Since both crosslinking and post-functionalizationutilize the same click reaction, PEGylation of the SCM could becatalyzed by the residual catalyst in the solution. Additional catalyst(e.g., CuCl₂ and/or sodium ascorbate) can optionally be added toincrease the speed of the reaction. Termination by propargyl alcohol isunnecessary in the post-functionalization because the surface of thenanoparticles is fully protected by the hydrophilic polymer. Afterreaction, excess 5 and other impurities were easily removed by dialysisagainst water. DLS revealed a significant increase in the particle sizeto about 100 nm in diameter, consistent with the attachment of the PEGchains.

Cleavage of the geminal diol groups enabled a determination of thedegree of functionalization in the PEG-SCMs. Integration of the methylprotons from the surfactant and PEG in the cleaved nanoparticlesindicated that, on average, one surfactant was functionalized with0.6-0.8 PEG chains (FIG. 6). The level of functionalization is in linewith the number of residual triple bond left on the alkynyl-SCM. If theaggregation number of 1 in the micelle is 50, about 30-40 PEG chainswould be present on the surface of a single nanoparticle. Consideringthe crowded environment that the PEG chains may experience on thesurface of an SCM, the level of post-functionalization is quiteremarkable. Dodecyltrimethyl ammonium bromide has a micellar aggregationnumber of 55 in water at 20° C. The aggregation number increases withlonger chain length and decreases with larger head group (see Rosen, M.J., Surfactants and Interfacial Phenomena, 2^(nd) Ed.; Wiley; New York,1989; p 16).

The SCMs have also been functionalized with a mannose derivative, 6, forits many interesting biological properties. The resulting particles hada hydrodynamic diameter of 25-40 nm, larger than the parent alkynyl-SCMsbut smaller than the PEG-SCMs. Periodate-digestion was not performedbecause the additional geminal diols on the sugar are incompatible withthe analysis.

Transmission microscopy allowed us to visualize the SCMs directly. Thesamples were stained with 2% phosphotungstic acid. The parentalkynyl-SCMs gave the smallest nanoparticles in the micrograph,averaging about 10 nm in diameter. The mannose-SCMs are larger, withtheir size mostly ranging from 15-25 nm. The size increase is reasonablewith the surface-functionalization. The PEG-SCMs are much larger,showing spherical particles mostly 40-60 nm in diameter and some aslarge as 80 nm (FIG. 7). Interestingly, the PEG-SCMs are positivelystained (i.e., particles appear dark) by phosphotungstic acid whereasthe alkynyl- and mannose-SCMs are negatively stained. For thefunctionalized mannose- and PEG-SCMs, the particle size determined fromTEM is smaller than that from DLS. The result is reasonable because DLSmeasures the hydrodynamic diameter of fully hydrated nanoparticles insolution whereas TEM measures the stained, dry particles in thecollapsed state.

Additional functional groups can be added by selecting from variousoptions for the crosslinker. For example, when azido ketone 2c wasemployed as the crosslinker, the nanoparticles gave both carbonyl (1745cm⁻¹) and triple bond (2111 cm⁻¹) stretches in the FT-IR spectroscopy,whereas the particles prepared with 2a only showed the triple bondstretch (2132 cm⁻¹).

In conclusion, the creation of a new platform for synthetic multivalentligands by surface-crosslinking of alkynylated surfactant micelles hasbeen described. The synthesis of the starting materials and thepreparation of the nanoparticles are simple. The click chemistry used inboth crosslinking and post-functionalization ensures unparalleledfunctional group compatibility and allows the final functionalizedmaterials to be prepared in a one-pot reaction at room temperature inwater. Additional functional groups (e.g., ketones) may be introducedthrough selection of the corresponding crosslinker, enablingpost-modifications orthogonal to the 1,3-dipolar cycloaddition. Thesefeatures represent significant advantages and cost benefits over othermultivalent platforms, such as dendrimers and gold nanoparticles, thattypically involve multistep synthesis or expensive metals. The SCMs areuseful in both chemical and biological applications. These particles canbe used for controlled release and delivery applications.

General Experimental Methods. ¹H and ¹³C NMR spectra were recorded on aBRUKER DRX-400 or on a VARIAN VXR-400 spectrometer. Dynamic lightscattering (DLS) was performed on a PD2000DLS^(PLUS) dynamic lightscattering detector. ESI-MS was performed on a FINNIGAN TSQ700 massspectrometer and MALDI-TOF mass was recorded on a ThermobioanalysisDynamo mass spectrometer. Fourier-Transform Infrared (FTIR) Spectra wererecorded on a BRUKER IFS 66V spectrometer. Transmission electronmicroscopy (TEM) studies were carried out on a PHILIPS CM 30 instrument,operating at 150 kV. The TEM samples were stained with 2%phosphotungstic acid (pH=6.2).

The preparation of compound 1 was carried out by methods analogous toknow procedures, as well as by a standard ammonium bromide formationprocedure (Scheme 1-2). For example, see Brun et al., Synthesis 2002,1385-1390; Percec et al., J. Am. Chem. Soc. 1998, 120, 8619-8631; andTanabe et al., Org. Lett. 2007, 9, 4271-4274.

4-(Dodecyloxy)benzaldebhyde. Potassium carbonate (24.8 g, 180 mmol) wasadded to a solution of 4-hydroxybenzaldehyde (3.66 g, 30 mmol) in DMF(90 mL) at room temperature. After the mixture was stirred at 60° C. for1 h, 1-bromooctane (8.7 mL, 36 mmol) was added slowly. The reactionmixture was stirred overnight at 80° C. under N₂, cooled to roomtemperature, and poured over 200 mL of icy water. The mixture wasextracted with EtOAc (3×50 mL). The combined organic phase was washedwith brine (100 mL), dried over MgSO₄, and concentrated in vacuo to givea yellow oil (13.67 g). ¹H NMR (400 MHz, CDCl₃, δ): 9.88 (s, 1H), 7.84(d, J=8.7 Hz, 2H), 7.00 (d, J=8.7 Hz, 2H), 4.06 (t, J=6.6 Hz, 2H),1.83-1.76 (m, 2H), 1.62-1.23 (m, 18H), 0.90 (t, J=8.8 Hz, 3H).

4-(Dodecyloxy)benzyl alcohol. A solution of 4-(dodecyloxy) benzaldehyde(10 g, 35 mmol) in THF (80 mL) was added dropwise to a stirredsuspension of NaBH₄ (2.7 g, 70 mmol) in dry THF (70 mL) at 0° C. Thereaction mixture was stirred at room temperature overnight and wasquenched with a small amount of water (5 mL). The solid formed wasremoved by filtration and the filtrate was concentrated in vacuo. Theresidue was dissolved in CHCl₃ (50 mL). The resulting solution waswashed with brine (3×20 mL), dried over anhydrous Na₂SO₄, andconcentrated in vacuo to give a white powder (9.56 g, 94%). ¹H NMR (400MHz, CDCl₃, δ): 7.29 (d, J=8.4 Hz, 2H), 6.90 (d, J=8.8 Hz, 2H), 4.62 (d,J=6.0 Hz, 2H), 3.97 (t, J=6.8 Hz; 2H), 1.79-1.51 (m, 2H), 1.51-1.26 (m,19H), 0.90 (t, J=6.4 Hz, 3H).

4-(Dodecyloxy)benzyl bromide. A solution of PBr₃ (2.0 mL, 20 mmol) inanhydrous CH₂Cl₂ (25 mL) was added slowly to a stirred solution of4-(dodecyloxy)benzyl alcohol (3.0 g, 10 mmol) in CH₂Cl₂ (50 mL) at 0° C.The reaction mixture was stirred for 3 h at room temperature and slowlypoured into a large amount of water (ca. 600 mL). The product wasextracted with CHCl₃ (3×50 mL). The combined organic phase was washedwith brine (3×20 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo to give a yellow solid (3.01 g, 90%). ¹H NMR (300 MHz, CDCl₃, δ):7.32 (d, J=6.6 Hz, 2H), 6.86 (d, J=6.6 Hz, 2H), 4.50 (s, 2H), 3.97 (t,J=6.6 Hz, 2H), 4.05-4.00 (m, 6H), 1.79-1.72 (m, 2H), 1.45-1.21 (m, 12H),0.90 (t, J=6.3 Hz, 3H).

4-(Dodecyloxy)benzyltripropargylammonium bromide (1). Tripropargylamine(0.85 mL, 9 mmol) in acetone (6 mL) was slowly added to a solution of4-(dodecyloxy)benzyl bromide (3.01 g, 8.5 mmol) in acetone (10 mL).After 3 d at room temperature, acetone was removed in vacuo and theresidue purified by column chromatography over silica gel withCH₂Cl₂/MeOH=20/1 to 10/1 as the eluents to give a white powder (2.1 g,61%). ¹H NMR (400 MHz, CDCl₃, δ): 7.56 (d, J=8.4 Hz, 2H), 7.08 (d, J=8.4Hz, 2H), 4.62 (s, 2H), 4.32 (s, 6H), 4.22 (s, 3H), 4.02 (t, J=6.4 Hz,2H), 1.73-1.68 (m, 2H), 1.40-1.24 (m, 18H), 0.87 (t, J=5.6 Hz, 3H); ¹³CNMR (100 MHz, CDCl₃, δ) 161.6, 134.2, 130.4, 117.2, 115.5, 114.7, 76.6,75.3, 68.3, 68.0, 63.5, 49.7, 41.9, 29.6, 29.4, 29.3, 29.1, 26, 22.7,14.1; ESI-MS (m/z): [M−Br]⁺ calcd for C₂₈H₄₀NO⁺ 406. found 406.

Crosslinkers 2a-c can be prepare by techniques analogous to thosedescribed by Glacon et al. (Carbohydr. Res. 2004, 23, 95-110); Haridaset al. (Org. Lett. 2008, 10, 1645-1647); and Dave et al. (TetrahedronLett. 2004, 45, 2159-2162), or as illustrated in Scheme 1-3 below.

Compound 2a. Sodium azide (2.52 g, 38.7 mmol) was added to a solution of2,2′-bioxirane (0.5 ml, 6.45 mmol) in water (10 mL). After 12 h at roomtemperature, the reaction mixture was extracted with ethyl ether (3×50mL). The combined organic phase was washed with brine (3×20 mL), driedover anhydrous Na₂SO₄, and concentrated in vacuo to give a colorless oil(0.75 g, 67%). ¹H NMR (400 MHz, D₂O, δ): 3.69-3.64 (m, 2H), 3.31 (d.J=5.6 Hz, 4H).

Compound 2b. Sodium azide (3.9 g, 60 mmol) was added to a solution ofp-xylylene dibromide (4.0 g, 15 mmol) in dry acetone (20 mL). Thereaction mixture was heated to reflux under N₂ for 12 h. The solid wasremoved by filtration and the filtrate was concentrated in vacuo to givea yellow oil (2.56 g, 91%). ¹H NMR (400 MHz, CDCl₃, δ): 7.35 (s, 4H),4.36 (s, 4H).

Compound 2c. Sodium azide (2.60 g, 40.0 mmol) was added to a solution of1,3-dichloroacetone (1.016 g, 8.0 mmol) in acetone (15 mL). The mixturewas stirred at room temperature for 12 h. The solid was removed byfiltration and the filtrate was concentrated in vacuo to give a yellowoil (1.075 g, 96%). ¹H NMR (400 MHz, CD₃Cl₃, δ): 4.09 (s, 4H).

Azido PEG compounds can be prepared using techniques analogous to thosedescribed by Rivera et al. (Can. J. Chem. 2003, 81, 1076-1082) andParrish et al. (J. Am. Chem. Soc. 2005, 127, 7404-7410), or asillustrated below in Scheme 1-4. The value of n in the repeating unit ofPEG can be such that the molecular weight of the PEG groups is about 100to about 10,000.

PEG-OTs. Poly(ethylene glycol) monomethyl ether (M.W. 2000, 4.0 g, 2.0mmol) and p-toluenesulfonyl chloride (0.76 g, 4.0 mmol) were dissolvedin dry CH₂Cl₂ (20 mL). Pyridine (0.33 mL, 2.0 mmol) was added under N₂.The reaction mixture was stirred at 25° C. under N₂ for 24 h. Afteraddition of 1 M HCl aqueous solution (10 mL), the mixture was extractedwith CH₂Cl₂ (3×20 mL). The combined organic phase was dried over MgSO₄,filtered, and concentrated in vacuo. The residue was purified by columnchromatography over silica gel with CH₂Cl₂/MeOH=10/1 as the eluent togive a white solid (4.3 g, 99%). ¹H NMR (400 MHz, CD₃Cl₃, δ): 7.80 (d,J=8 Hz, 2H), 7.35 (d, J=8 Hz, 2H), 3.82-3.45 (m, 172H), 3.37 (s, 3H).

Compound 5. Sodium azide (0.39 g, 6.0 mmol) was added to a solution ofPEG-OTs (4.3 g, 2.0 mmol) in DMF (40 mL). The reaction mixture wasstirred at 80° C. for 12 h, diluted with water (200 mL), and extractedwith ethyl ether (3×50 mL). The combined organic phase was washed withwater (3×30 mL) and concentrated in vacuo to give a white solid (3.51 g,86%). ¹H NMR (400 MHz, CD₃Cl₃, δ): 3.82-3.38 (m, 172H); 3.37 (s, 3H).

Modification of saccharides can be carried out by the methods of Watt etal. (Org. Biomol. Chem. 2005, 3, 1982-1992); Hayes et al. (Tetrahedron2003, 59, 7983-7996); Rivera et al. (Can. J. Chem. 2003, 81, 1076-1082),and Kleinert et al. (Eur. J. Org. Chem. 2004, 18, 3931-3940); or asillustrated in Scheme 1-5 below.

1,2,3,4,6-Penta-O-acetyl-D-mannopyranoside. Sulfuric acid (2 drops) wasadded to a stirred mixture of D-mannose (4.98 g, 27.8 mmol) and aceticanhydride (27 mL) at 0° C. The reaction mixture was allowed to warm toroom temperature after 10 min. After 30 min, the mixture was poured intoicy water (100 mL) and extracted with EtOAc (100 mL). The organic layerwas washed with water (3×100 mL) and saturated NaHCO₃ aqueous solution(3×50 mL), dried over MgSO₄, and concentrated in vacuo to give a yellowoil (10.0 g, 93%). The product was used in the next step without furtherpurification.

2′-Bromoethyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside. Borontrifluoride etherate (5.1 ml, 40.0 mmol) was added to a solution of1,2,3,4,6-penta-Oacetyl-D-mannopyranoside (3.9 g, 10.0 mmol) and2-bromoethanol (0.82 ml, 11.0 mmol) in dry CH₂Cl₂ (40 mL). The reactionmixture was stirred in dark under N₂ for 3 h, diluted with CH₂Cl₂ (50mL), and quenched by saturated NaHCO₃ aqueous solution (100 mL). Theorganic phase was washed with water (3×50 mL), dried over MgSO₄, andconcentrated in vacuo. The residue was purified by column chromatographyover silica gel with ethyl acetate/hexane=2/1 as the eluent to give awhite powder (1.81 g, 40%). ¹H NMR (400 MHz, CD₃Cl₃, δ): 5.33-5.26 (m,3H), 4.87 (1H, s), 4.29-4.25 (m, 1H), 4.15-4.11 (m, 2H), 3.99-3.95 (m,1H), 3.90-3.86 (m, 1H), 3.53 (t, J=8 Hz; 2H), 2.16 (s, 3H), 2.11 (s,3H), 2.05 (s, 3H), 2.00 (s, 3H).

2′-Azidoethyl-2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside. Sodium azide(1.625 g, 25 mmol) was added to a solution of 2′-bromoethyl2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (1.81 g, 4.0 mmol) inanhydrous DMF (10 mL). The reaction mixture was stirred at 60° C.overnight. DMF was removed by rotary evaporation and the residue wasdissolved in CH₂Cl₂ (100 mL). The CH₂Cl₂ solution was washed with water(4×50 mL), dried over MgSO₄, filtered, and concentrated in vacuo to givea white powder (1.72 g, 94%). ¹H NMR (400 MHz, CD₃Cl₃, δ): 5.37-5.27 (m,3H), 4.87 (s, 1H), 4.30-4.27 (m, 1H), 4.14-4.10 (m, 1H), 4.01-4.03 (m,1H), 3.90-3.84 (m, 1H), 3.69-3.64 (m, 1H), 3.52-3.41 (m, 2H), 2.16 (s,3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H).

Compound 6. Sodium methoxide (0.1 N, 0.82 mL, 0.082 mmol) in methanolwas added to a solution of 2′-azidoethyl 2,3,4,6tetra-O-acetyl-α-D-mannopyranoside (1.72 g, 4.1 mmol) in dry methanol(20 mL). After 2 h at room temperature, acidic ion-exchange resin (Dowex50wx8-200) was added until pH=6-7. The resin was removed by filtrationand the filtrate was concentrated in vacuo to give a white powder (1.0g, 98%). ¹H NMR (400 MHz, D₂O, δ): 4.91 (d, J=1.6 Hz, 1H), 3.98-3.97 (m,1H), 3.93-3.88 (m, 2H), 3.88-3.82 (m, 1H), 3.78-3.65 (m, 4H), 3.54-3.48(m, 2H).

Preparation of Alkynyl-SCMs. Compound 2a (3.45 mg, 0.02 mmol), CuCl₂ (10μL of 6.7 mg/mL aqueous solution, 0.5 μmol), and sodium ascorbate (10 μLof 99 mg/mL aqueous solution, 5 μmol) were added to a micellar solutionof 1 (10 9.73 mg, 0.02 mmol) in millipore water (2.0 mL). The reactionmixture was stirred slowly at room temperature for 24 h before propargylalcohol (0.6 μL) was added. After another 12 h, the mixture was dialyzedagainst deionized water using a 500 Da molecular weight cut-off tubing.

Preparation of PEG-SCMs. Alkynyl-SCMs were prepared as above. Aftercrosslinking, (without the addition of propargyl alcohol) 5 (81.0 mg,0.04 mmol), CuCl₂ (10 μL of 6.7 mg/mL aqueous solution, 0.5 μmol), andsodium ascorbate (10 μL of 99 mg/mL aqueous solution, 5 mmol) wereadded. After another 12 h at room temperature, the mixture was dialyzedagainst deionized water using a 6-8 kDa molecular weight cut-off tubing.

Preparation of Mannose-SCMs. Alkynyl-SCMs were prepared as above. Aftercrosslinking, (without the addition of propargyl alcohol), 6 (15 mg,0.06 mmol), CuCl₂ (10 μL of 6.7 mg/mL aqueous solution, 0.5 μmol), andsodium ascorbate (10 μL of 99 mg/mL aqueous solution, 5 μmol) wereadded. After another 12 h at room temperature, the mixture was dialyzedagainst deionized water using a 500 Da molecular weight cut-off tubing.

Synthesis of Alkynyl-Keto SCMs. Compound 2c (2.8 mg, 0.02 mmol), CuCl₂(10 μL of 6.7 mg/mL aqueous solution, 0.5 μmol), and sodium ascorbate(10 μL of 99 mg/mL aqueous solution, 5 μmol) were added to a micellarsolution of 1 (9.73 mg, 0.02 mmol) in millipore water (2.0 mL). Thereaction mixture was stirred slowly at room temperature for 24 h beforepropargyl alcohol (0.6 μL) was added. After another 12 h, the mixturewas dialyzed against deionized water using a 500 Da molecular weightcut-off tubing.

Example 2 Facile Preparation of Organic Nanoparticles by InterfacialCrosslinking of Reversed Micelles and Templated Synthesis ofSub-nanometer Au-Pt Nanoparticles

Surfactants can self-assemble into a rich array of ordered phasesdepending on their molecular structures, temperature, and amounts ofpolar, nonpolar, and surfactant ingredients. There has been along-standing interest in capturing these noncovalently stabilizedphases by covalent bonds to prepare ordered nanomaterials. Covalentfixing of these self-assembled phases not only enhances their stabilitybut also enables them to be used as templates for further materialsynthesis.

Reversed micelles (RMs) are formed when a small amount of water is addedto a mixture of a suitable surfactant in nonpolar solvent(s). RMs arewidely employed as media for catalysis and templates to prepareinorganic nanomaterials. Nevertheless, the dimension of the inorganicmaterials obtained from the templated synthesis rarely correlates withthe size of the RM templates. Collisions of these dynamic assembliesresult in coalescence as well as rapid exchange of the entrapped waterand dissolved contents, making it difficult to predict the size andmorphology of the final materials. Although covalent fixing of the RMsmight be expected to circumvent these problems, it is exactly the sameproblems one has to face when attempting to capture RMs of the originalsize. Earlier attempts to polymerize RMs produced objects much largerthan the original assemblies. Recently, McQuade and co-workers reportedthe first example of capturing RMs by free radical polymerization (J.Am. Chem. Soc. 2003, 125, 5351-5355). A key to their success was thedesign of an AOT-like surfactant with two polymerizable groups near thehead group.

This Example provides a method to use highly efficient thiol-ene “click”reactions to crosslink RMs exclusively at the interface. The synthesisof the starting materials and the crosslinking can be readily carriedout using standard synthetic techniques. These interfacially crosslinkedreversed micelles (ICRMs) can be used to template the synthesis of bothsmall (3-4 nm) and ultrasmall (<1 nm) metal nanoparticles. Size controlis controllable, based on the amounts of the metal precursor versussurfactant employed in the synthesis. Nanoalloys are obtained by simplycombining two metal precursors in the same reaction. These features arethe direct results of the covalent nature of the templates, which aredifficult to obtain from conventional RMs.

Quatemary ammonium surfactant 10 (4-(dodecyloxy)benzyl-triallylammoniumbromide) was prepared by alkylation of 4-hydroxybenzaldehyde with1-bromododecane, reduction of the aldehyde with sodium borohydride,bromination of the resulting alcohol with phosphorus tribromide, andnucleophilic displacement of the bromide with triallyl amine. Opticallyclear RM solutions were obtained in a 2:1 heptane/chloroform mixturewith W₀<15 (W₀=[H₂O]/[1]). In FIG. 8, dithiol crosslinker 9 iscrosslinker 2. Although RMs could form without chloroform, this solventaided the dissolution of dithiol crosslinker 2 in the nonpolar mixture.

Crosslinking of the RMs was achieved by UV-irradiation of the solution(W₀=5) in the presence of 2,2′-dimethoxy-2-phenylacetophenone, aphotoinitiator. After crosslinking, the sharp peaks in the ¹H NMRspectrum of a 2:3 mixture of 10 and 2 (FIG. 8a ) became broad and thealkenic protons (H_(c)) of 10 and those on the crosslinker disappearedalmost completely (FIG. 8b ). After evaporation of solvents and washingwith water, the materials obtained were soluble in common organicsolvents such as chloroform, tetrahydrofuran, and acetone, but insolublein water and methanol. Notably, the methyl and methylene protons of thedodecyl chain were sharp and well-resolved, whereas the protons near theammonium head group were broad and weak or absent (FIG. 8c ). Theseresults are consistent with crosslinking at the interface in the RMconfiguration, which constrains the movement of the ammonium head groupbut not that of the hydrocarbon tail pointing outward.

The CRMs were additionally characterized by dynamic light scattering(DLS) and transmission electron microscopy (TEM). Although the CRMs weresoluble in many organic solvents (e.g., chloroform, methylene chloride,THF, acetone, butanone, DMF, DMSO), their sizes determined by DLS werequite different. TABLE 1 summarizes the hydrodynamic diameters of theRMs and ICRMs determined in various solvents. The RMs were captured in2:1 heptane/CHCl₃. Instead of trying to obtain the refractive index andviscosity for the mixed solvent, the diameter of the RMs was calculatedusing the parameters of heptane (entry 1) and CHCl₃ (entry 2),respectively. The sizes (5-6 nm) obtained were indeed very close andcompared favorably with those of conventional AOT RMs (2-3 nm),considering the (longer) dodecyl tail of 1 and the extra phenylenespacer. FIG. 9 shows the size-distribution of these nanoparticles inseveral organic solvents. The average hydrodynamic radius was 16 nm inboth acetone (FIG. 9a ) and butanone (FIG. 9c ) for freshly madesamples. Over time (about 1-2 hours), however, the scattered lightintensity increased significantly for the acetone sample and largeparticles, hundreds of nanometers in radius, started to form (FIG. 9b ,data collected at 40 minutes).

TABLE 1 Hydrodynamic Diameters of RMs and ICRMs Determined by DLS^(a)Sample Solvent Diameter (nm) RMs of Compound 1 2:1 heptane/CHCl₃ 6^(b)RMs of Compound 1 2:1 heptane/CHCl₃ 5^(c) ICRMs of Compound 1 CHCl₃190   ICRMs of Compound 1 THF 13   RMs of Compound 2 2:1 heptane/CHCl₃5^(b) RMs of Compound 2 2:1 heptane/CHCl₃ 4^(c) ICRMs of Compound 2CHCl₃ 5  ICRMs of Compound 2 THF 4  ^(a)The diameters were averages offive measurements. Each measurement was based on 20 accumulations ofdata collection. The relative standard deviations within the fivemeasurements ranged from 1 to 9%. ^(b)The diameter was calculated usingthe viscosity and refreactive index of heptane. ^(c)The diameter wascalculated using the viscosity and refractive index of CHCl₃.

These large particles eventually became insoluble in acetone andprecipitated out of the solution, at which time the scattered lightintensity became extremely low (data not shown). These changes can beattributed to particle aggregation in the relatively polar acetonebecause the size stayed constant (about 16-20 nm) in the less polarbutanone indefinitely (FIG. 9c ). The size was different again inchloroform, about 95 nm in radius, and stayed unchanged over time (FIG.9d ). The particles were smallest in THF, only about 7 nm in radius,although some very large particle existed (FIG. 9e ). Because largeparticles scatter light substantially more than small particles, thesmall particles were actually the dominant species, as shown by themass-normalized size distribution (FIG. 9f ).

Because it appeared that aggregation could be significant in somesolvents, TEM was used for further characterization of the CRMs.Transmission microscopy (TEM) allowed for visualization of the CRMsdirectly. Both small particles (R≈10 nm) and large particles (R=15-20nm) could be found in a sample prepared from THF (FIG. 10a ). Thesesizes were quite consistent with those obtained by DLS. It is possiblethat the large particles were simply aggregates of the small particles,as suggested by the DLS study. Alternatively, the large particles couldresult from coalescence of small particles during crosslinking and thuscould be permanent. To further confirm the covalent capture,phosphotungstic acid-stained CRMs were examined, which displayedimproved contrast under TEM (FIG. 10b ). The TEM images showed manyspherical particles 10-15 nm in radius. Interestingly, some largerparticles, e.g., the one at upper right corner and the one near thelower right corner, were observed to form by aggregation.

With the introverted ammonium groups at the core, the ICRMs can extractanionic metal precursors, such as tetrachloroaurate (AuCl₄ ⁻), fromwater to chloroform. Addition of sodium borohydride to the chloroformsolution immediately turned its color from light yellow to purple. Thechoice of the crosslinker was important to the RM capture. Althoughorganic-soluble materials could be obtained when a hydrophobiccrosslinker such as 1,4-butanedithiol was used in the photocrosslinking,the materials were unsuitable for the templated synthesis and only bulkgold precipitate formed following the same procedures.

When equivalent amounts of aurate and surfactant 10 were used (i.e.,[AuCl₄ ⁻]/[10]=1), the UV-vis spectrum showed broad absorption with apeak at 520 nm (FIG. 11), indicating the formation of goldnanoparticles >2 nm in diameter. The solution was stable indefinitely inour hands, showing no signs of precipitation and/or aggregation. Thesurface plasmon absorption band at 520 nm became significantly weakerand a higher-energy peak appeared at about 330 nm when the [AuCl₄⁻]/[10] was reduced to 0.1, indicating formation of both large (>2 nm)and ultrasmall (<1 nm) nanoparticles under this condition. The amount ofultrasmall nanoparticles increased even more as the [AuCl₄ ⁻]/[10] ratiowas reduced to 0.02. The absorption at 330 nm became stronger and thatat 520 disappeared completely. These data indicate that the size of thegold nanoparticles was controlled by the amount of aurate used in thetemplated synthesis. Different amounts of water (W₀=1 or 5), forexample, caused very little difference in the position of the absorption(FIG. 11).

TEM analysis gave results consistent with those from the UV-visspectroscopy analysis. The particles obtained at [AuCl₄ ⁻]/[10]=1averaged about 3 nm. Similar sized nanoparticles were also observed at[AuCl₄ ⁻]/[10]=0.1, but far fewer particles appeared in the micrographeven though the concentration of the sample was higher than what wasused for [AuCl₄ ⁻]/[10]=1. Presumably, subnanometer gold particlesexisted in sample at [AuCl₄ ⁻]/[10]=0.1, but were undetectable by TEM.This trend continued as [AuCl₄ ⁻]/[10] was reduced to 0.02. Even fewerparticles were observable in TEM and what could be seen were very small(1 nm or less).

Subnanometer gold and silver clusters have attracted interest in recentyears as novel biolabels and optoelectronic emitters. As their sizeapproaches the Fermi wavelength of electrons, noble metal clustersdisplay dramatically different optical, electronic, and chemicalproperties, from either the bulk or the nanoscale metals. Thephotoluminescence of these materials agreed completely with the UV-visand TEM data. Under a hand-held UV lamp (365 nm), the Au-ICRMs preparedwith [AuCl₄ ⁻]/[10]=1 gave no signs of luminescence. As the [AuCl₄⁻]/[10] ratio decreased, the sample became increasingly fluorescent,with the sample prepared with the lowest aurate giving the brightestblue light.

A unique property of subnanometer gold clusters is their atom-likeproperties. Indeed, the excitation/emission spectra of these Au-CRMsresembled those of molecular fluorophores, displaying the maximum at315/354 nm. The electronic transition energy of Au clusters is known toscale with inverse cluster radius. The excitation/emission wavelengthsfor Au₃, Au₄, Au₅ clusters, for example, were reported to be 305/340,313/371, and 330/385 nm, respectively. Comparison with the literaturedata suggests that the dominant fluorescent species in our Au-ICRMs ismost likely Au, clusters, a reasonable result based on the [AuCl₄ ⁻]/[1]ratio of 0.02 and typical aggregation number of RMs.

Thus, nanoalloys such as Au—Pt, were readily prepared via the templatedsynthesis described herein. Using PtCl₆ ²⁻ or a 1:1 mixture of AuCl₄ ⁻and PtCl₆ ²⁻ with 2 mol % of the total metal precursor, fluorescent Ptand Au—Pt clusters were obtained following similar procedures (FIGS. 12and 13). The excitation/emission wavelengths of the Pt clusters were346/399 nm. Interestingly, the Au—Pt clusters displayed an intermediatevalues (329/390 nm). The few metal atoms appeared to indeed exist asalloys instead of separate clusters.

In summary, a simple method to crosslink RMs at the interface using thehighly efficient thiol-ene click reaction has been described. The methodallowed covalent fixing of the dynamic surfactant assemblies andproduced stable organic nanoparticles soluble in common solvents. Thesecrosslinked RMs can be used to produce both nanometer and subnanometermetal particles by varying the ratio of metal precursor/surfactant usedin the synthesis. Although dendrimers can also be suitable templates forsubnanometer gold clusters, such reaction often take several days tocomplete, and post-purification (e.g., centrifugation) is needed toremove large particles formed during the synthesis. The straightforwardsynthesis of 10, the simplicity of the templated synthesis, and the manyapplications of the noble metal clusters in photonics and catalysis makethe ICRMs highly attractive templates in advanced nanomaterialssynthesis.

Experimental Details.

Synthesis of 10: Triallylamine (0.70 mL, 4.0 mmol) in acetone (2 mL) wasslowly added to a solution of 4-(dodecyloxy)benzyl bromide (0.71 g, 2.0mmol) in acetone (3 mL). After 3 d at room temperature, acetone wasremoved by rotary evaporation and the residue purified by columnchromatography over silica gel with CH₂Cl₂/MeOH=20/1 to 10/1 as theeluents to give a white powder (0.63 g, 64%). ¹H NMR (400 MHz, CDCl₃,δ): 7.58 (d, J=8.4 Hz, 2H), 6.95 (d, J=8.8 Hz, 2H), 6.04-5.96 (m, 3H),5.78-5.30 (m, 6H), 4.91 (s, 2H), 4.18 (d, J=7.2 Hz, 6H), 3.98 (t, J=6.4Hz, 2H), 1.81-1.75 (m, 2H), 1.45-1.26 (m, 18H), 0.89 (t, J=6.8 Hz, 3H);¹³C NMR (100 MHz, CDCl₃, δ) 160.74, 134.34, 128.31, 125.06, 118.64,114.95, 68.07, 63.58, 61.49, 31.74, 29.50, 29.47, 29.44, 29.41, 29.25,29.18, 29.0, 25.87, 22.52, 22.27, 13.98; ESI-MS (m/z): [M−Br]⁺ calcd forC₂₈H₄₆NO⁺412. found 412.

Preparation of CRMs: Water (1.8 μL) was added to a solution of 10 (9.8mg, 0.02 mmol) in heptane (1.0 ml) and CHCl₃ (0.5 ml). The mixture washand shaken and sonicated at room temperature for 1 min to give anoptically clear solution. After addition of 2 (4.6 mg, 0.03 mmol) and2,2′-dimethoxy-2-phenylacetophenone (25.6 mg/mL in chloroform, 10 μL,0.1 μmol), the mixture was irradiated in a Rayonet photoreactor for ca.10 h until most alkenic protons in 10 were consumed. The organicsolvents were removed by rotary evaporation and the residue was washedby water to give a white power (11.2 mg).

Preparation of Au-CRM: A 5 mM aqueous solution of HAuCl₄ (2 mL) wasadded to a 10 mM RCM solution in chloroform (2 mL). The aqueous phasebecame colorless and the organic phase turned yellow upon stirring. Afreshly prepared aqueous solution of sodium borohydride (0.2 M, 1 mL)was slowly to the vigorously stirred reaction mixture. The organic phaseturned purple immediately and the color intensified over 2 h. Theorganic phase was washed with water three times and concentrated byrotary evaporation. The residue could be redissolved in common organicsolvents and were stable over a period of several months.

Example 3 Reversible Crosslinkers

The SCMs and CRMs described herein can also be prepared with otherreversible crosslinkers, such as diazido compounds 3 and 4.

Preparation of bis(2-azidoethyl)disulfide (3):

Preparation of bis-(2-hydroxy ethyl) disulfide (B): Aqueous sodiumbicarbonate (10%, 40 mL) was added to a solution of 2-mercaptoethanol(A, 3.0 mL, 43 mmol) in dichloromethane (40 mL). A solution of bromine(2.0 mL, 39 mmol) in dichloromethane (10 mL) was slowly added while themixture was stirred at 0° C. After addition, the organic phase wasseparated and the aqueous phase was extracted with dichloromethane (3×10mL). The organic solvent was evaporated and the residual was purified bycolumn chromatography (CH₂Cl₂:CH₃OH=3:1) to afford B as a colorless oil(0.34 g, 13%). ¹H NMR (400 MHz; D₂O, δ): 3.81 (t, J=6.4 Hz, 4H), 2.85(t, J=6.4 Hz, 4H).

Preparation of bis-(2-bromoethyl)disulfide (C): Concentrated H₂SO₄ (10mL) was slowly added to a stirred 48% HBr aqueous solution (14 mL) at 0°C. Compound B (0.33 g) was added dropwise to the above mixture. After 24hours at room temperature, the mixture was heated on a steam bath for 3hours. Dichloromethane (10 mL) was added to the cooled reaction mixture.The organic layer was separated, washed with water and 10% Na₂CO₃aqueous solution, dried over anhydrous Na₂SO₄, filtered, andconcentrated to give compound C (0.48 g, 86%). ¹H NMR (400 MHz, D₂O, δ):3.63 (t, J=8 Hz, 4H), 3.12 (t, J=8 Hz, 4H).

Preparation of bis(2-azidoethyl)disulfide (3): Compound C (0.226 g, 0.95mmol) was dissolved in DMF. To this solution was added sodium azide(0.31 g, 4.75 mmol) and the mixture was stirred at 80° C. for 10 hours.The product was extracted with ethyl ether three times. The combinedorganic layers were washed with brine, and dried over anhydrous Na₂SO₄.After filtration and evaporation, the product was dried under vacuum togive 3-1 as a yellow oil (0.162 g, 84%). ¹H NMR (400 MHz, CDCl₃, δ):3.62 (t, J=6.8 Hz, 4H), 2.89 (t, J=6.8 Hz, 4H). See Rai et al., Bioorg.Med. Chem., 2008, 16, 7301-7309.

Preparation of 1-(bis(2-azidoethoxy)methyl)-4-methoxybenzene (4):

Preparation of 1-(bis(2-chloroethoxy)methyl)-4-methoxybenzene:2-Chloroethanol (8.05 mL, 120 mmol), 4-methoxybenzaldehyde (6.07 mL, 50mmol), benzene (25 mL), and p-toluenesulfonic acid (8.6 mg, 0.05 mmol)were combined and the mixture was heated to reflux. The water formed wasremoved by a Dean-Stark trap. When no additional water appeared in theDean-Stark trap, the reaction mixture was cooled to room temperature anda solution of sodium methoxide (0.1 g) in 2 mL of methanol was addedrapidly under stirring. The mixture was diluted with hexane, washed withbrine, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo.The crude product was used in the next step without furtherpurification.

Preparation of 1-(bis(2-azidoethoxy)methyl)-4-methoxybenzene (4): Crude1-(bis(2-chloroethoxy)methyl)-4-methoxybenzene (5.8 g) was dissolved inDMF. Sodium azide (13.5 g, 200 mmol) was added and the reaction mixturewas stirred at 80° C. for 10 hours. The product was extracted withhexane three times. The combined organic layers were washed with brine,and dried over anhydrous Na₂SO₄. After filtration and evaporation, theproduct was dried under vacuum to give 4 as an oil. ¹H NMR spectroscopyindicated that the oil was a 1:1 mixture of product and4-methoxybenzaldehyde.

Example 4 Rapid Release of Entrapped Contents fromMulti-Functionalizable, Surface Crosslinked Micelles

It was surprisingly discovery that the SCMs can release entrappedcontents extremely rapidly (<1 minute) upon cleavage of thecrosslinkages. Because of the unparalleled tolerance of the clickreaction to functional groups, SCMs were able to be prepared with avariety of crosslinkers and, as a result, different environmentalstimuli can be used to trigger the release of the entrapped contents(“cargo”). This method combines the simplicity of physical entrapmentwith stimuli-triggered release of entrapped contents, making the SCMshighly useful in the delivery and controlled release of hydrophobicdrugs.

Pyrene was used as a surrogate for a hydrophobic drug because of itsenvironmentally sensitive fluorescence. Pyrene has five vibronic bandsthat respond to environmental polarity differently. The intensity ratiobetween the third (˜384 nm) and the first band (˜372 nm), in particular,is sensitive to changes in the environment. As illustrated in FIG. 14,the emission spectrum of pyrene varies with the concentration ofamphiphile 1. Changes in I₃/I₁ indicate that pyrene is in a morehydrophobic microenvironment in 1 mM aqueous solution of amphiphile 1than in 0.05 mM. The CMC of the surfactant is about 1.4×10⁻⁴ M accordingto surface tension measurement and 1.5×10⁻⁴ M, according to FIG. 15.

Pyrene-containing SCMs were then prepared according to (FIG. 22),following similar procedures for the synthesis of SCMs described above.Briefly, an aqueous solution containing 38 μM pyrene and 10 mM ofsurfactant 1 was prepared. Because the solubility limit of pyrene inwater is 0.67 μM, the majority of the dissolved pyrene resided withinthe surfactant micelles. Addition of a crosslinker (e.g., one or more ofcompounds 2-4) and CuCl₂/sodium ascorbate initiated the crosslinking andthe reaction was allowed to continue for 12-24 hours at roomtemperature.

Entrapment of pyrene was confirmed by fluorescence spectroscopy. Whenthe pyrene-containing SCMs were diluted by water so that theconcentration of crosslinked 1 was below its CMC, I₃/I₁ of pyreneremained unchanged at 0.84-0.85 (the value above the CMC, see FIG. 15)for a period of more than six months. However, as soon as periodic acid(HIO₄) was added to the mixture to cleave the 1,2-diol group in thecrosslinker, I₃/I₁ dropped quickly (FIG. 14). Remarkably, release of thepyrene was so rapid that, by the time HIO₄ was added and the solutionwas mixed by gentle vortexing (<1 minute), the change in I₃/I₁ wascomplete.

The end I₃/I₁ value was somewhat dependent on the amount of HIO₄ added.One equivalent of the cleaving agent (2×10⁻⁵ M) reduced the I₃/I₁ to0.74-0.75, higher than the 0.70 observed in the uncrosslinked surfactantbelow the CMC (FIG. 15). Some of the 1,2-diol groups may have beenuncleaved under the latter conditions. The addition of 10 and 100 equivof HIO₄ apparently disintegrated the SCMs quickly and completely (FIG.16), as the final the I₃/I₁ was similar or even slightly lower than 0.70in the uncrosslinked micelles. Incidentally, periodic acid at highconcentration (2×10⁻³ M) was found to quench the fluorescence of pyrenesignificantly, which could explain why the I₃/I₁ ratio was even lowerthat the 0.70 observed for pyrene below the CMC of surfactant 1.

The outstanding tolerance of the click reaction allowed for theincorporation of other stimuli-sensitive crosslinkers in the SCMs.Diazide 3, for example, contains a disulfide bond that can be cleaved bythiols such as dithiol 5. Release of pyrene was once again found tooccur extremely fast after addition of 5 (FIG. 17). Excess 5 was notneeded to reduce the I₃/I₁ ratio to 0.70 and 1 equiv of dithiol 5 wasenough to completely release the entrapped pyrene.

Cleaving of 1,2-diol and disulfide bonds in crosslinked polymers wasreported to take hours to days to complete and often require millimolarconcentrations of reducing thiols (Sun et al., Biomacromolecules 2006,7, 2871; Koo et al., Chem. Comm. 2008, 6570; Zhang et al.,Biomacromolecules 2008, 9, 3321). In contrast, the SCMs expelled pyreneextremely rapidly. Considering the concentration (2×10⁻⁵ M) of thesurfactant (in the pyrene-containing SCMs) and the releasing agent (HIO₄or 5), the release was remarkably efficient. The electrostatic stress ofthe system likely speeds up the cleaving reaction compared to polymermicelle systems, resulting in rapid stimuli-triggered release by theSCMs.

Pyrene-containing the SCMs with acetal-containing 4 as the crosslinkerwere also prepared to evaluate the release of pyrene under acidicconditions because acid-triggered release is important in many deliveryapplications. For example, endosomes and liposomes are well known to bemore acidic than the cytosols (Mellman et al., Annu. Rev. Biochem. 1986,55, 663). Successful delivery by endocytosis often requiresacid-triggered release, and cancerous and inflammatory tissues are alsoknown to be more acidic than normal tissues (Helmlinger et al., Clin.Cancer Res. 2002, 8, 1284).

Initially, pyrene-containing SCMs prepared using acetal 4 showed nochange in fluorescence at pH=5 over a period of 96 hours. The result wasinitially a surprise because the SCMs were surrounded by acidic waterand the p-methoxybenzyl acetal group in 4 is highly prone to hydrolysis.The compound, for example, can undergo partial hydrolysis during waterworkup.

Concerned that pyrene fluorescence did not directly monitor changes tothe nanoparticles, dynamic light scattering (DLS) was employed. DLScorrelates the scattered light with the diffusion coefficient ofparticulate species in the sample. FIG. 18 illustrates the percentchange in the intensity of scattered light for variouspyrene-containing-SCMs. Disintegration of the nanoparticles was onceagain found to be extremely rapid for SCMs prepared with 2 or 3 as thecrosslinker (Δ and □, respectively). The intensity of scattered lightdropped to 15 and 24% of the original value, respectively, when 1equivalent of HIO₄ and dithiol 5 were added to the corresponding SCMs.

The change in light intensity was clearly slower for the acid-triggereddisassembly. The nanoparticles prepared with acetal 4 as the crosslinkerdisintegrated gradually over the period of 60 minutes at 37° C.,although the majority of the change occurred in the first 20 minutes.The intensity of scattered light dropped to 15% of the original valueafter 48-96 hours. In contrast, the nanoparticles at pH=7 displayednegligible changes in scattered light over 96 hours. A hydrogelcrosslinked with a similar p-methoxybenzyl acetal was reported torelease entrapped protein over several hours instead of 20 minutes(Murthy et al., J. Am. Chem. Soc. 2002, 124, 12398).

In summary, it was found that the SCMs could eject entrapped hydrophobiccontents extremely rapidly upon cleavage of the surface crosslinkers. Asindicated by Scheme 4-1 and demonstrated by the examples above,multivalent surface functionalization of these nanoparticles is readilyaccomplished by addition of azide-functionalized polymers and ligands. Acombination of crosslinkers may be used and the acid- orredox-sensitivity of the SCMs can be tailored for specific applications.

This simple method combines the ease of physical entrapment and thepreciseness of chemical ligation and therefore does not require anycovalent modification of the entrapped agents. With the additionalbenefits of multivalent surface modification, tuning of surface charge(e.g., by using anionic, nonionic, or zwitterionic surfactants withmultiple alkynyl groups), and outstanding tolerance of the clickreaction for functional groups, the SCMs can be useful materials foractive agent delivery and controlled release.

Example 5 Preparation of Phospholipid Vesicles by Extrusion

To prepare phospholipid vesicles by extrusion, phospholipids are firstsuspended in a buffered saline solution to give large, multilamellarvesicles. The vesicles are then repeatedly passed through apolycarbonate filter with 100 nm pores. The result is uniformly sized,unilamellar vesicles (large unilamellar vesicles, or LUV), approximately100 nm in diameter. A LIPOSOFAST extruder from Avestin, Inc. (Ottawa.Ontario, Canada) is suitable for the extrusion. The LIPOSOFAST extruderis a syringe-based membrane extruder that is inexpensive and easy touse, and it allows one to prepare 0.5 to 1 mL batches of phospholipidsat a time. Another suitable vesicle extruder, the Mini-Extruder, isavailable from Avanti Polar Lipids (Alabaster, Ala.).

Note that the phospholipids must be handled at a temperature above theirtransition temperature (Tc) from gel to liquid crystalline phase. Thenatural phospholipids are generally used, which are in the liquidcrystal phase at room temperature. If other types of phospholipids areused, it may be necessary to carry out the preparatory procedures at atemperature above the Tc (not necessarily at room temperature).Alternative procedures to make unilamellar vesicles include sonication,detergent/dialysis and detergent/Bio-Beads.

Materials and Solutions: HEPES buffered saline (HBS): 100 mM NaCl; 20 mMHepes/NaOH buffer, pH 7.5; 0.02% (w/v) sodium azide. Alternatively, 50mM Tris buffer, pH 7.5, may be substituted for the Hepes buffer. The HBSshould be stored at room temperature.

Phospholipid Stock Solutions Phospholipid name Concentration MW PCL-alpha-Phosphatidylcholine, egg 10 or 25 mg/mL 761 PSL-alpha-Phosphatidylserine, 10 mg/mL 810 bovine liver-Na salt PEL-alpha-Phosphatidylethanolamine, 10 mg/mL 768 bovine liver

Phospholipid Stock Solutions can be purchased from commercial supplierssuch as Avanti Polar Lipids (Alabaster, Ala.), dissolved in chloroform.Stock solutions should be stored at −20° C. under argon and should notbe stored for more than 3 months (6 months for PC).

Methods:

1. Dispense 2.6 μmole total phospholipids (PL) in a glass test tube (a13×100 mm tube is a convenient size). Examples of amounts of PL to usein making PCPS or PCPSPE vesicles include the following:

For PC:PS vesicles (80:20 molar ratio) 63 μL PC (at 25 mg/mL) =1.58 mg=2.08 μmole (or 158 μL at 10 mg/mL) 42:L PS (at 10 mg/mL) =0.42 mg =0.52μmole For PC:PE:PS vesicles (40:40:20 molar ratio) 32:L PC (at 25 mg/mL)=0.79 mg =1.04 μmole (or 79 μL at 10 mg/mL) 80:L PE (at 10 mg/mL) =0.80mg =1.04 μmole 42:L PS (at 10 mg/mL) =0.42 mg =0.52 μmoleThe contents of the stock vials of phospholipid should be overlaid withargon gas before capping and returning them to the freezer.

2. In the fume hood, dry the PL mixture under a gentle stream ofnitrogen or argon. When dry, speed-vac for an additional 1 hour toovernight under high vacuum, to remove any residual chloroform.

3. To the dried-down PL, add 2.6 mL room temperature HBS solution andcover the end of the tube, such as with parafilm. Incubate 1 hour atroom temperature with intermittent agitation.

4. Vortex tube vigorously to completely resuspend the PL. The resultshould be a milky, uniform suspension. Freeze and thaw the suspensionthree to five times. For example, freeze in dry ice/alcohol bath; thawrapidly at 37° C.

5. Clean the LIPOSOFAST device with ethanol and dry it well. Assemblethe device with two membranes held between the two “O” rings and filtersupports according to the manufacturer's directions. Two polycarbonatemembranes with 100 nm pore size are generally suitable, although otherpore sizes can also be used.

6. Load 0.5 mL of the lipid suspension into one of the two glasssyringes and attach it to the Luer lock on one side of the device. Closethe other (empty) syringe and attach it to the Luer lock on the oppositeside of the device.

7. Press the loaded syringe to pass its entire contents through thefilter and into the opposing syringe. Repeat this process alternatelywith the two syringes for a total of at least about 11 passes. It isimportant that an odd number of passes are employed, so that the finalproduct ends up in what was originally the empty syringe. This willensure that none of the starting multilamellar vesicles will contaminatethe final product. In addition, it is important that this procedure beperformed at a temperature that is above the Tc for your lipid mixture.

8. Remove the final product and repeat steps 6 and 7 for the remaining,unprocessed phospholipid suspension, until all of the suspension hasbeen processed.

9. Store the final product at 4° C. The result is a uniform suspensionof unilamellar vesicles (about 100 nm in diameter) containing a total of1 mM phospholipid in HBS.

The final phospholipid concentration can be confirmed by assaying totalphosphorus content. Additional description and techniques are describedby

Mui et al., Extrusion technique to generate liposomes of defined size;Methods Enzymol. (2003) 367:3-14. See also

http://www.avantilipids.com/extruder.html;

http://www.avantilipids.com/ExtruderAssembly.html; and

http://www.avantilipids.com/LUVET.html.

Example 6 Crosslinked Organic Particles for Catalysis

Diminishing natural resources, deteriorating environmental conditions,and rising green-house gases in the atmosphere have placed greatchallenges for creating efficient catalytic processes. Development ofenergy-efficient and environmentally benign catalysis is important to asustainable chemical industry. Due to its abundance, nonflammability,and nontoxicity, water is an attractive solvent for green chemicaltransformations. Although a number of organic reactions are beingcarried out in water on industrial scales, expanding the scope ofaqueous-based industrial organic reactions requires fundamentally newconcepts in catalytic technology.

This example provides methods to expand aqueous biphasic catalysis usinghydrophobic organometallic catalysts entrapped within crosslinkedmicelles. The surface-crosslinked micelles (SCMs) can improve masstransfer and product separation of organic reactions in comparison toconventional technologies for aqueous biphasic catalysis.

Background. Aqueous biphasic catalysis is an attractive process fortransition-metal catalyzed organic reactions. Water, as a green solvent,is abundant, nonflammable, and nontoxic. The high heat capacity of wateris beneficial to the temperature control of a reaction. Recovery andrecycling of the catalyst, as well as separation of products, arestraightforward when they reside in two immiscible phases. In addition,water can speed up certain organic reactions by hydrophobic effects.Indeed, aqueous-based catalysis is being used in a number of industrialorganic transformations, including hydroformylation of propene tobutyraldehyde, hydrodimerization of butadiene, and the Wacker oxidation.

Expanding the scope of aqueous biphasic catalysis, however, facessignificant challenges. Some approaches to aqueous-based organicreactions using transition metal catalysts attach water-soluble groups(e.g., sulfonate, sulfate, ammonium, carboxylate, phosphate, orhydroxyl) or polymers to the metal-binding ligands. Researchers havealso anchored catalysts on insoluble solid supports such as silica. Overthe last decades, a large number of ligands have been designed andsynthesized, allowing aqueous-based catalysis successfully performed onthe lab scale for a growing number of reactions, e.g., hydroformylation,hydrogenation, olefin polymerization, olefin metathesis, cross-coupling,and catalytic oxidation.

Scaling up these aqueous reactions to the industrial scale, however,remains highly challenging. Pulling transition metal catalysts intowater, in fact, is the easier part of the problem. Getting the organicreactant to come in contact with the water-soluble catalyst is typicallymore challenging. Although there are methods to improve the solubilityof organics in water, e.g., by adding water-miscible organic co-solventor surfactants, these methods inevitably compromise product isolation.The presence of organic solvent will render both the reactant andproduct more soluble. Because of the high heat capacity of water,separation of the (water-miscible) organic solvent is energy-intensiveand organic solvent-containing water is no longer nontoxic and createsan environmental hazard. Although surfactants can promote dissolution oforganic molecules in water, their surface activity promotes emulsionformation, which is a significant problem in a chemical process in whichgood phase separation is important for product purification.

This example describes a method to trap unmodified or largely unmodifiedtransition metal catalysts in highly crosslinked micelles. The overallstructures have striking resemblance to enzymes in the sense that thecatalytic site is located within a hydrophobic microenvironmentsuspended in the aqueous phase. The effective concentration of thesubstrate is enhanced by the hydrophobic micelle and mass transfer isfacilitated by the fast exchange of organic reactant/product frommicelle to micelle (and to the organic bulk phase). The straightforwardsynthesis of the crosslinkable surfactants and SCMs, as well as theability of using unmodified or largely unmodified conventional ligandsfor the transition metal catalysts, makes the method practical andapplicable to large industrial reactions.

The examples above describe a simple method to crosslink surfactantmicelles. The reaction design includes, for example, a highlyalkynylated surfactant 1, synthesized in a few simple steps frominexpensive, commercially available starting materials. Highly efficientclick reactions (i.e., 1,3-dipolar cycloaddition between a terminalalkyne and an azide) were used for crosslinking, which occurs at roomtemperature in the presence of an azide-containing crosslinker (e.g.,1,4-diazidobutane-2,3-diol (2a)) and Cu(I) catalyst above the CMC of 1,1.5×10⁻⁴ M in water (FIG. 23). Although hydrophobic crosslinkers such as1,4-bis(azidomethyl)benzene (2b) or 1,3-diazidopropan-2-one (2c) may beused as well, water-soluble crosslinkers such as 2a gave suitableresults. The resulting nanoparticles were about 8-10 nm in diameteraccording to dynamic light scattering (DLS) and about 10 nm by TEM.

(1) Hydrophobic Organometallic Catalysts Entrapped within SCMs. Aone-step synthesis of SCMs using the highly efficient click chemistry isdiscussed above. High surface-crosslinking density enabled physicalentrapment of small hydrophobic guests (e.g., pyrene or drug molecules)within the core of the micelle even when the sample was diluted belowthe CMC of the surfactant. Water-soluble catalytic nanoparticles can beprepared by physically or chemically trapping phosphine- andsalen-complexed transition metal catalysts inside the SCMs. The micellarenvironment not only allows direct solubilization of unmodified orlargely unmodified hydrophobic catalysts in water but also can greatlyfacilitate mass transfer between the water-insoluble organic reactantand the catalyst in the aqueous phase. By covalently crosslinking thesurfactants in the micellar configuration, the surface activity of thesurfactant is eliminated and emulsion formation, a serious plague forthe separation of products in traditional surfactant-assistedorganometallic catalysis, can be avoided. Cooperative catalysis andsite-isolation can be implemented, further improving the catalyticefficiency and selectivity.

(2) Increased Hydrophobic Free Volume within SCMs. The packing densityof the hydrocarbon tails within the SCMs can be decreased, therebyproviding increased voids or “hydrophobic free volume”. These voids aretypically currently filled with water but would readily accept organiccompounds. In aqueous biphasic catalysis, the hydrophobic free volume ofan SCM increases its affinity for the organic reactant. Three strategiescan be used to decrease the hydrophobic packing density of the SCMs: (a)using crosslinkable surfactants with sacrificial hydrophobes, (b) usingcrosslinkable surfactants with removable, noncovalent or reversiblecovalent linkages, and/or (c) using uncrosslinkable diluents (organicsolvent or amphiphiles). With larger hydrophobic free volume inside theSCM, the effective concentration of the substrate increases near thetransition metal catalyst, thereby improving catalytic efficiency.

By trapping organometallic catalysts in crosslinked micelles, the needfor ligand-modification for catalysts is eliminated and the approachavoids the dichotomy derived in conventional aqueous-based biphasiccatalysis, i.e., efficient mass transfer between the reactant organicphase and the water-soluble catalyst prefers maximal miscibility of theorganic and the aqueous phase, but efficient product separation prefersminimal miscibility.

These two approaches are complementary and synergistic. Suitableorganometallic catalysts can be identified by the first approach and canbe used to optimize key parameters such as surfactant structure,crosslinking density, catalyst loading, and reaction conditions. Thesecond approach can be performed independent of the first, usingfluorescent probe to monitor the hydrophobic free volume of the SCMs.The data obtained can be applied to catalysis directly to improvesubstrate binding and mass transfer. Application of this technology willexpand the scope of aqueous based catalytic organic reactions.

Hydrophobic Organometallic Catalysts Entrapped within SCMs. Thecrosslinking method can be used to prepare water-soluble SCMs withphosphine- or salen-complexed transition metal catalysts trapped inside,for use with industrially important reactions such as hydrogenation,hydroformylation, and olefin epoxidation. Other reactions (e.g.,palladium-catalyzed cross-coupling) may also be carried out using theseprocedures. The micellar environment not only allows solubilization ofunmodified or largely unmodified hydrophobic catalysts in water but alsogreatly facilitate the mass transfer between the water-insoluble organicreactant and the catalyst. Crosslinking enables site isolation of thecatalyst and may shut off certain catalyst decomposition/deactivationpathways. The catalyst can also be used at higher concentrations in theaqueous phase, thereby increasing the reaction rates. For catalysts thatbenefit from cooperative interactions of two or multiple metal centers,two or multiple catalysts can be trapped in the same SCM. The micellarbody itself, with its surface charges, can also be useful in enhancingthe catalytic efficiency.

Design and Preparation of SCM-Encapsulated Catalysts. Phosphine is anextremely versatile ligand in organometallic catalysis. Industriallyimportant reactions such as hydrogenation, hydroformylation, andcross-coupling may be catalyzed by phosphine-complexed transitionmetals. Scheme 6-2 lists several commercially available phosphineligands that can be entrap within the SCMs.

Preparation of a SCM-encapsulated phosphine-rhodium catalyst is shown byFIG. 19. The procedure is adapted from the pyrene-entrapment discussedabove. As an example, a hydrophobic phosphine (e.g., Xanphos) can befirst protected by borane in a well-established procedure (Brunel etal., Phosphane-boranes: synthesis, characterization and syntheticapplications. Coordin Chem Rev 1998, 180, 665-698). Protection isimportant because phosphines (especially alkyl substituted) can beoxidized in air and readily react with azides via the Staudingerreaction. Protection with borane eliminates these problems and enablesthe phosphine-borane complex to be included inside the SCMs similarly topyrene. After crosslinking, treatment with an amine (e.g., Et₂NH) cangenerate the free phosphine inside the SCM. The active catalyst can beprepared by combining the phosphine-containing SCMs and aRh(l)-precursor, e.g., Rh(COD)₂BF₄. The biphasic catalysis is shownschematically by the two-layer mixture in FIG. 19. The top phasecontains the water-insoluble organic reactant/product and thecatalyst-containing SCMs are located in the bottom aqueous phase. Rapidstirring is important to phase-separated reactions.

(1) As shown in FIG. 19, the catalyst is located within a hydrophobic(top) micro-environment even though the surrounding SCM is in theaqueous (bottom) phase. This arrangement differs from conventionalbiphasic catalysis in which the catalyst is surrounded by water. Whereasthe organic reactant will have difficulty) approaching the catalyst inthe conventional method, it can easily get into the hydrophobic micelleto come in contact with the catalyst. Because both the reactant and thecatalyst are confined in a nanosized microenvironment, significant rateacceleration may be observed due to the higher effective concentrationof the substrate near the catalyst.

Conventional micelles are dynamic assemblies of individual surfactants.For ionic surfactants, the typical residence time for a surfactantwithin a micelle is on the order of 10⁻⁶-10⁻⁷ s. In an SCM, thesurfactants are fixed by covalent bonds but the reactant and product arefree to move about. Given the fast exchange of surfactant betweenmicelles, even if the exchange of reactant is several orders ofmagnitude slower, it can still be a bottleneck to most organicreactions. If a surfactant with 12 to 16 carbons can move from onemicelle to another quickly, organic molecules with fewer carbons shouldbe able to do similarly because the barrier (i.e., exposure ofhydrophobic surface to water) for a reactant/product to migrate from oneSCM to another is the same as that for a surfactant to jump from onemicelle to another.

(2) Surfactants have been used in aqueous-based organic reactions tosolubilize the reactant and/or the catalyst. However, they promoteemulsion formation between the organic and aqueous components and createenormous problems in product separation. Alternatively, one can enhancethe solubility of the reactant by adding a water-miscible organicco-solvent, but the increased solubility is achieved at the expense ofproduct separation and water-contamination. In our described method, thesurfactants are fixed by crosslinking in the micellar configuration.Because the SCMs are completely hydrophilic on the exterior, thematerials are not believed to have surface activity. In this way, fastmass transfer and good product separation can be achieved at the sametime.

(3) The catalysts are encapsulated within the SCMs and isolated from oneanother. Site isolation may not only avoid certain pathways (e.g.,dimerization or bridging) for catalyst decomposition, but can also allowtheir use at relatively high concentrations in the aqueous phase, bothof which can speed up the reaction. Cascade reactions are achievablewhen different SCM-entrapped catalysts are present at the same time.Such reactions are frequently found in biological systems (e.g.,metabolism) but are often hampered by catalyst incompatibility.

(4) Certain catalysis reactions (e.g., epoxide hydrolysis catalyzed byCo-Salen complexes) benefits from cooperativity between two metalcenters. The methods described herein permit simultaneous trapping oftwo or multiple catalysts within an SCM. In typical homogeneouscatalysis, the encounter between the reactant and the catalyst iscollision-based. The time of encounter can be significantly shorter thanin the SCM-based catalysis, where the organic reactant may stay in thesame SCM for longer period of time, thereby increasing cooperativity.

(5) The affinity between the SCM and the organic reactant can be tunedin a rational fashion. As the affinity of the SCM for the reactant isincreased, one can increase the effective concentration of the substratenear the catalyst.

(6) As shown by FIG. 19, preparation of the encapsulated catalystsinvolves simple synthesis with commercially available ligands. Thistranslates to lower costs in the production of the catalysts. Withexcellent mass transfer and easy product separation, the method isapplicable to large industrial reactions.

Alternative Embodiments. Many transition metals require more than onephosphine ligand for stabilization. The Wilkinson catalyst[RhCl(PPh₃)₃], which catalyzes homogeneous hydrogenation of unhinderedalkenes, needs at least two phosphines in the active form. A simple wayto achieve this is to trap a bisphosphine ligand such as Diphos insidethe SCM. Another strategy is to employ commercially availableacid-functionalized phosphine 7 (Scheme 6-2 above). With electrostaticinteractions between the carboxylate form of 7 and cationic 1, one caninclude multiple molecules of compound 7 within an SCM. Thetriarylphophine can be located inside the micelle instead of in theaqueous phase due to its hydrophobicity.

Most ligands discussed herein are large in comparison to common organicreactants and products. If a large ligand/catalyst can be physicallytrapped within the SCM without other adverse effects (e.g., in masstransfer), physical entrapment can be achieved. Pyrene trapped insidethe SCMs stayed inside the micelle for more than 6 months when the SCMwas diluted below the CMC of 1 and pyrene concentration was well belowits solubility in water, indicating physical entrapment. In someembodiments, the crosslinking density for physical entrapment of ahydrophobic ligand/catalyst may be higher than the initially preparedparticles described above. The organic reactant and product can pass inand out of the SCM. Thus, untrapped ligands/catalysts may be extractedand may leach into the organic phase. Two strategies can be used toaddress these issues.

(a) The crosslinking density of the SCMs can be increased. Instead ofbis-azide such as 2a, tris-azide 8 or 9 can be used as a crosslinker.Data indicates that water-solubility is helpful to the crosslinking. Insome embodiments, compound 9 can provide improved crosslinking thancompound 8, which is less polar. Surfactant 10 can also be used as acrosslinker. Being very similar in structure, it can form mixed micelleswith 1, which can be readily crosslinked to form highly crosslinkedSCMs.

Triphenylphosphine-containing (or pyrene-containing) SCMs with differentcrosslinkers can be used to evaluate crosslinking density. The SCMs canbe dried and stirred in a large amount of organic solvent (e.g., CHCl₃).The concentration of triphenylphosphine (or pyrene) can be determined byappropriate techniques (NMR or fluorescence) to identify potentialleaching. Soxhlet extraction can also be used for evaluatingcrosslinking density. If the ligand/guest molecule within the SCMs cansurvive Soxhlet extraction using a range of solvent, e.g., EtOH, CHCl₃,hexane, the chance of leaching will be minimal.

(b) Covalent fixation of the ligand. Physical entrapment allowscommercially available hydrophobic ligands such as Diphos, Xanphos, orBINAP to be directly used. Nonetheless, too high a crosslinking densitymay be detrimental if relative large reactants/products are involvedand/or high mass transfer is critical. In some cases, it may bepreferable to keep the crosslinking density relatively low while havingthe ligand covalently attached to the SCMs. Azide-functionalizedphosphine-borane complexes 11-13 can be readily prepared using standardtechniques All three ligands can be prepared in two to three simplesteps from commercially available phosphines. The azide group allowstheir covalent attachment to the SCMs during crosslinking. As before,the borane can be removed by an organic base after the SCM preparation.

Organic Reactions Catalyzed by SCM-Containing Phosphine-ComplexedTransitions Metals. A range of phosphine-containing SCMs can beprepared. Crosslinking density, mono- or bi-dentancy, physical orchemical entrapment, chirality (e.g., Diphos vs. BINAP), and bite angle(e.g., Diphos vs. Xanphos) can be varied during preparation. To evaluatethe effectiveness of SCM-encapsulated catalysts, two industriallyimportant reactions can be analyzed

(a) Rh-Catalyzed Hydrogenation. The reaction can be performed understandard literature conditions (see Joo, Aqueous biphasichydrogenations; Accounts Chem Res 2002, 35, (9), 738-745). Briefly, thephosphine-containing SCMs and a Rh(I)-precursor, such as Rh(COD)₂BF₄,can be mixed in water to form the catalyst. The reaction can be carriedout at room temperature and followed by NMR or GC analysis. Scheme 6-3lists examples of the substrates that can be used in the biphasichydrogenation. The results can then be compared with homogeneouscatalysis using the same amount of catalyst. The benefit of biphasiccatalysis is the ease of product separation and catalyst recovery. Thereaction can be accelerated by higher pressure of hydrogen and/or largerconcentration of catalyst.

Relatively hydrophilic substrates can be used in the reactions. Allylalcohol, for example, is miscible with water. When hydrophilicitydecreases from allyl alcohol to 3-buten-1-ol and then to geraniol (14),the reactivity can be closely monitored to determine relativereactivity. It Aromatic hydrocarbons are less hydrophobic thancorresponding aliphatic compounds. Since 1 has an aromatic moiety in thestructure, the aliphatic and aromatic olefins can have differentaffinities for the SCM. Finally, prochiral substrates such as 15 and 16can be evaluated by these reactions. Micelles can increase theenantiomeric selectivity of transition metal catalysts and the SCMs canhave a similar effect.

(b) Rh-Catalyzed Hydroformylation. Hydroformylation is an importantindustrial process because heterogeneous catalysis is challenging forthis reaction (unlike hydrogenation). Water-soluble sulfonatedphosphines have been used successfully for hydroformylation of lowolefins. However, as the chain length increases, mass transfer becomesinefficient and the reaction rate becomes unacceptable. Hydroformylationof linear olefins (e.g., 1-pentene through 1-dodecene) can be performedwith phosphine-containing SCMs. These olefins can enter the SCMs and beconverted. As long as the reactant and product can quickly exchange withthose in the bulk organic phase, reasonable reactivity can bemaintained. Bite angle is known to be important to the activity of theRh catalyst. Xanphos, for example, is particularly active. Thelinear/branched selectivity in these hydroformylation can be evaluatedusing the SCMs described herein.

Metallosalen Catalysts Encapsulated within SCMs. Metallosalens catalyzea wide variety of reactions including epoxidation, epoxide hydrolysis,cyclopropanation, and aziridation. These additional reactions and theirunique features can open up new strategies in the catalyst design andallow testing of novel catalytic concepts. Chart 3 lists severalmetallosalens to be studied. Their sizes are comparable to thephosphines shown in Chart 2; thus the strategies mentioned above (i.e.,higher crosslinking density and covalent fixation) should be useful fortrapping the catalysts inside the SCMs. Unlike the phosphines,salen-metal complexes are compatible with the click chemistry and may bedirectly incorporated.

Two reactions catalyzed by metallosalen complexes—catalytic epoxidationof olefins by Mn(III)-salen (17) and epoxide hydrolysis⁵¹⁻⁵² usingCo(III)-salen (18 or 19) can be carried out using the particlesdescribed herein. Catalyst 17 is extremely effective at asymmetricalepoxidation of olefins. For the catalytic epoxidation, establishedsubstrates such as (Z)-prop-1-enylbenzene and cis-methyl cinnamate canbe used to evaluate the biphasic catalysis. The reaction rates andenantiomeric selectivity can be compared with those in homogeneousreactions.

The cheapest and most effective oxidant for the metallosalencomplexes—catalytic epoxidation of olefins is bleach (NaClO). Becausethe SCMs are positively charged on the surface, the hypochlorite (ClO⁻)anion can be concentrated on the SCM surface. A rate acceleration,unavailable in a homogeneous system, may result. Also, the activecatalyst is an oxo-Mn(V)-salen complex and tends to form unreactivep-oxo-Mn(IV) dimers unless an additive such as 4-phenylpyridine N-oxideor N-methylmorpholine N-oxide is present. The catalyst of interest canbe trapped inside the SCM. If the micelle/catalyst ratio is high duringthe crosslinking, a single catalyst within an SCM (although some SCMsmay be empty) can be confirmed and can avoid the additive. Although anadditive may not be a problem on a laboratory scale, its elimination inan industrial reaction represents a significant improvement,particularly for separating the product by decantation (as in biphasiccatalysis).

One issue with of concern with epoxidation reactions is the strongoxidizing abilities of the oxo-Mn(V) species formed during thecatalysis. Under certain conditions, the hydrocarbon chain of thecrosslinked 1 may be oxidized over time. Oxidation may not be a problemwhen a large excess of the olefin substrate is present. However, anypotential oxidation of the SCMs will affect the catalyst lifetime andrecycling may become problematic. A solution to this potential problemis the preparation of a fluorocarbon version of the crosslinkablesurfactant, e.g., 20. The Mn-salen complex inside the SCM in this casewill be essentially in a Teflon-lined nanoreactor, which is highlystable toward oxidation.

Co(III)-salen complexes is efficient at catalyzing kinetic resolution ofepoxide by hydrolyzing one enantiomer of a chiral epoxide faster thanthe other. The catalysis is known to operate through cooperation betweentwo metallosalen complexes, with one activating the epoxide and theother water. The same strategies to incorporate multiple phosphines maybe used with the methods described herein. For example, catalyst 18 hasan anionic carboxylate; its electrostatic interactions with the cationicsurfactant (1) can allow multiple Co-salen complexes to be included.

Another strategy to include two salen catalysts within a single SCM isto use a dicarboxylate (⁻OOC—X—COO⁻) as a ligand for the Co(III)species. Catalyst 19a (Scheme 6-4) can be prepared by ligand exchange ofa monomeric Co(III)-salen complexes with a dicarboxylic acid. After itsincorporation inside the SCMs, the dicarboxylate can be removed using alarge excess of acetate. Such a step is important because the epoxideand the water molecule on opposite faces of the Co-Salen dimer will notbe able to react easily. In the unlikely event that acetate is unable todisplace the dicarboxylate, 19a can be used, which contains a germinaldiol group. The group can be readily cleaved by periodate, generatingtwo Co(III)-salen complexes from the dimer.

Site isolation of catalyst is an important factor in the methodsdescribed herein. Site isolation of the catalysts can allow for cascadereactions catalyzed by two or more catalysts. The conversion of olefinsto diols through the epoxide intermediate can therefore be evaluated.Because both Mn(III)-salen and Co(III)-salen catalysts can be prepared,the two SCM-encapsulated catalysts can be combined to performepoxidation and hydrolysis in one pot. Chiral Co(III)-salen is no longerneeded in this case, as the epoxide intermediate will be chiral from theasymmetric epoxidation.

Increasing Hydrophobic Free Volume within SCMs. Ionic surfactants formmicelles in a compromise between the hydrophobic interactions among thetails and the electrostatic interactions among the head groups. Asignificant amount of water can be present within the micellar core. Infact, ionic micelles are known to be “wetter” than nonionic micelles dueto the strong repulsion among the head groups. The “hydrophobic freevolume” of the SCMs can be systematically increased, thereby decreasingthe packing density of the hydrophobic tails inside the SCMs. In theabsence of organic reactant, these voids or hydrophobic free volume aretypically filled with water, but placing water in a hydrophobicmicroenvironment is energetically unfavorable. As soon as such a(catalyst-containing) SCM is placed in a mixture containing organicreactant, the affinity hydrophobic free volume for the organic reactantdrives the change. To increase the hydrophobic free volume of SCM, apolarity-sensitive fluorescent probe can be used to monitor thewater-content inside the SCM.

Methods to Detect the Hydrophobic Free Volume Inside SCMs. Dansyl hasbeen widely used as a polarity-sensitive fluorescent probe. Whenincorporated inside micelles, dansyl has been shown to be able toindicate the “wetness” of micelles. Ionic micelles, for example, havebeen demonstrated by dansyl probes to be “wetter” or more polar in theinterior than nonionic micelles. To include a dansyl group in theinterior of an SCM, an azide-containing dansyl derivative (e.g., 21) canbe used, which can be synthesized from commercially available materialsin two simple steps. Because of its hydrophobicity, 21 can besolubilized in the interior of the micelles and covalently crosslinkedwith 1 during the click-crosslinking.

The emission wavelength, emission intensity, and its accessibility by asmall organic quencher (acrylamide) are three indicators for thehydrophobic free volume of a dansyl-containing SCM. In general, dansyl'semission wavelength moves to the red and its emission intensitydecreases as its environment becomes more polar. When the micellar coreof an SCM has a larger hydrophobic volume or contains more water, thedansyl probe can decrease in intensity and shift to red in emission. Atthe same time, if acrylamide is placed in the solution, the smallorganic quencher can enter the SCM and favorably displace the watermolecules. Higher quenching is thus expected for a wetter SCM.

The hydrophobic free volume of an SCM can be increased with threedifferent but complimentary methodologies:

Using Crosslinkable Surfactants with Sacrificial Hydrophobes.Surfactants 22 and 23 are similar to 1 in having three alkynyl groupsfor crosslinking through the click reaction. Instead of an ether linkagebetween the phenyl and the hydrocarbon tail, these two surfactantscontain cleavable linkages such as ester (in 22) and siloxane (in 23).Micellization and crosslinking can be performed with these surfactantsas before, except that the hydrocarbon tails can be removed by esterhydrolysis and fluoride treatment, respectively, after surfacecrosslinking. In some embodiments, an SCM can be prepared with 22 or 23as the only surfactant after the hydrocarbon tails are removed. Coreremoval yields an empty nanocapsule. Without the hydrophobicinteractions from the tails, such a structure can be unstable unless theshell is highly crosslinked. Enhanced crosslinking density can stabilizethese particles. Both ¹H NMR spectroscopy and TEM can be used tocharacterize such structures. The dansyl group is connected to the SCMthrough a sulfamide group, which is resistant to hydrolysis. As long asit is not removed during core-removal, its fluorescence can indicate anincrease in polarity upon core removal.

Compounds 22 and 23 can be useful but are not necessary in thepreparation of a catalyst-containing SCM. Mixing a “permanent”surfactant (e.g., 1) and a sacrificial surfactant (e.g., 22 or 23) givesSCMs with different amounts of removal hydrocarbon tails. Once the esteror siloxane linkages are cleaved, SCMs that remain hydrophobic insidecan be obtained but that have different hydrophobic free volumes. Evenif the catalyst is covalently attached to the SCM and leaching is not aconcern, crosslinking density is important in maintaining the micellarconfiguration of the SCM.

Using Crosslinkable Surfactants with Noncovalent or Reversible CovalentLinkages. Surfactant 26 can be formed using the guanidinium carboxylatesalt bridge between 24 and 25. Although the hydrogen-bonded salt bridgeis weak in water, it is very strong near the lipid-water and air-waterinterface. Micelle cores can be significantly hydrophobicmicroenvironments and hydrogen-bonded complexes have been shown to bestable in micelles. By using noncovalent surfactants such as 26 to formthe SCM, the core removal can be much easier, e.g., washing with polarorganic solvent such as methanol, and changing the hydrocarbon tail ormixing and matching multiple tails will not involve different syntheses.

Imines have been used to as the linkage to form micelle-forming,reversible surfactants. The advantage of using a reversible covalentlinkage is that the hydrocarbon tails can be easily exchanged. Mixingand matching is also possible using several different amine and aldehydeprecursors. This method allows one to functionalize the interior of theSCMs. Once the imine is hydrolyzed, the aldehyde groups can be used asanchors for additional functional groups. Reduction amination, forexample, can easily place desired functional groups in the SCM core.

Using Noncrosslinkable Surfactants or Organic Additives in the SCMPreparation. Discussed above, the sacrificial surfactants arecrosslinked in the head groups with the permanent surfactant. Anoncrosslinkable surfactant such as 29, or even an organic additive suchas toluene or xylene, can also be used during the SCM preparation.Surfactants 1 and 29 can form mixed micelles, which can be crosslinkedthrough 1. Washing with methanol or dialysis can remove theuncrosslinked 29 from the SCM.

The surface crosslinking density of the SCM can be lower than those ofthe SCMs prepared as described above because part of the micellarsurface can be occupied by the noncrosslinkable trimethylammonium. Thisapproach is complimentary to those described above and can afford SCMswith quite different surface structure. Removal of 29 can create “pores”on the SCM surface, which can be useful to mass transfer in thecatalysis.

Mixing a surfactant with an organic additive, such as toluene or xylene,is known to afford swollen micelles. “Swollen” SCMs can result if 1 anda small amount of an organic additive are used in the SCM preparation.Catalyst-containing SCMs prepared in this fashion obviate the need tohydrolyze tails or to wash the noncovalently linked tail. These SCMs maybe used directly in the biphasic catalysis. The organic additive can beexchanged by the organic reactant, present in large excess. Anotherbenefit of this approach is that, although the SCM has an increasedhydrophobic free volume, the volume is occupied by hydrophobic molecules(first by the organic additive, then by the organic reactant) andinstability of SCM caused by unsatisfied hydrophobic interactions is nota concern.

Example 7 Pharmaceutical Dosage Forms

The following formulations illustrate representative pharmaceuticaldosage forms that may be used for the therapeutic or prophylacticadministration of a composition described herein, (hereinafter referredto as ‘Composition X’, wherein the composition includes a drug ordiagnostic agent):

(i) Tablet 1 mg/tablet ‘Composition X’ 100.0 Lactose 77.5 Povidone 15.0Croscarmellose sodium 12.0 Microcrystalline cellulose 92.5 Magnesiumstearate 3.0 Total 300.0 (ii) Tablet 2 mg/tablet ‘Composition X’ 20.0Microcrystalline cellulose 410.0 Starch 50.0 Sodium starch glycolate15.0 Magnesium stearate 5.0 Total 500.0 (iii) Capsule mg/capsule‘Composition X’ 10.0 Colloidal silicon dioxide 1.5 Lactose 465.5Pregelatinized starch 120.0 Magnesium stearate 3.0 Total 600.0 (iv)Injection 1 (1 mg/mL) mg/mL ‘Composition X’ 1.0 Dibasic sodium phosphate12.0 Monobasic sodium phosphate 0.7 Sodium chloride 4.5 1.0N Sodiumhydroxide solution q.s. (pH adjustment to 7.0-7.5) Water for injectionq.s. ad 1 mL (v) Injection 2 (10 mg/mL) mg/mL ‘Composition X’ 10.0Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1 Polyethyleneglycol 400 200.0 0.1N Sodium hydroxide solution q.s. (pH adjustment to7.0-7.5) Water for injection q.s. ad 1 mL (vi) Aerosol mg/can‘Composition X’ 20 Oleic acid 10 Trichloromonofluoromethane 5,000Dichlorodifluoromethane 10,000 Dichlorotetrafluoroethane 5,000

These formulations may be prepared by conventional procedures well knownin the pharmaceutical art. It will be appreciated that the abovepharmaceutical compositions may be varied according to well-knownpharmaceutical techniques to accommodate differing amounts and types ofactive ingredient ‘Composition X’. Aerosol formulation (vi) may be usedin conjunction with a standard, metered dose aerosol dispenser.Additionally, the specific ingredients and proportions are forillustrative purposes. Ingredients may be exchanged for suitableequivalents and proportions may be varied, according to the desiredproperties of the dosage form of interest.

Additional Embodiments:

-   1. An organic particle comprising surface crosslinked non-polymeric    organic amphiphiles;

wherein polar head groups of the amphiphiles are covalently crosslinkedto each other at the surface of the particle through triazole groups orthioether groups, and tail groups of the amphiphiles are arranged towardthe interior of the particle; and the particle is water-soluble.

-   2. The organic particle of embodiment 1 wherein the tail groups of    the amphiphiles arranged toward the interior of the particle are    non-polar hydrocarbon or fluorocarbon tail groups, polar tail    groups, or a combination thereof.-   3. The organic particle of embodiment 2 wherein the non-polar tail    groups of the amphiphiles comprise one or more (C₆-C₂₂) alkyl    groups, (C₆-C₂₂) fluoroalkyl groups, or a combination thereof, and    wherein the non-polar tail groups optionally comprise one or more of    ester, imine, boronate, disulfide groups or salt bridges linking a    portion of the tail to a head group of the amphiphile or another    portion of the tail that is linked to the head group of the    amphiphile.-   4. The organic particle of any one of embodiments 1-3 wherein the    tail groups of the amphiphiles are non-polar and the particle has a    hydrophobic core and a hydrophilic exterior; or the tail groups of    the amphiphiles are polar and the particle has a hydrophilic core    and a hydrophilic exterior.-   5. The organic particle any one of embodiments 1-4 wherein the    particle is in the form of a liposome comprising a bilayer of    amphiphiles, and the bilayer comprises one or more water    compartments between the bilayer of amphiphiles.-   6. The organic particle of any one of embodiments 1-5 wherein the    particle comprises one or more cargo molecules within the particle    or at the surface of the particle.-   7. The organic particle of embodiment 6 wherein the cargo molecules    comprise one or more of a drug, an organic nanoparticle, an    inorganic nanoparticle, a fluorophore, a diagnostic agent, and a    catalysts.-   8. The organic particle of any one of embodiments 1-7 wherein the    surface of the particle comprises one or more surface groups    comprising water-soluble polymers, fluorophores, biological ligands,    nucleic acids, nucleic acid analogues, catalysts, or a combination    thereof.-   9. The organic particle of embodiment 8 wherein the surface groups    are receptors or ligands for corresponding ligands or receptors on a    biological host.-   10. The organic particle of any one of embodiments 1-9 wherein the    surface crosslinking can be cleaved by heat, by a change in pH, by a    reducing agent, or by a combination thereof.-   11. A delivery system comprising a plurality of particles of any one    of embodiments 6-10, and a pharmaceutically acceptable diluent or    carrier.-   12. An organic particle comprising non-polymeric crosslinked    amphiphiles;

wherein the amphiphiles comprise one or more nonpolar alkyl orfluoroalkyl chains and one or more polar head groups;

the nonpolar chains are located on the exterior of the particle and thepolar head groups are oriented toward the interior of the particle; and

the amphiphiles are covalently crosslinked to each other near the headgroups through triazole groups or thioether groups.

-   13. The organic particle of embodiment 12 wherein the particle    comprises one or more metal salts or metal particles within the    organic particle.-   14. The organic particle of embodiment 12 or 13 wherein the particle    comprises one or more catalytically active groups oriented toward    the interior of the particle.-   15. A method for preparing a surface-crosslinked organic particle    comprising:

combining a plurality of non-polymeric amphiphiles and water to form anoncovalently associated self-assembled micellar structure;

wherein the non-polymeric amphiphiles have polar head groups andnon-polar tail groups, and the polar head groups comprise two or morealkynyl groups or azido groups;

combining the self-assembled structure with a plurality of crosslinkingagents, wherein the crosslinking agents comprise two or more azidogroups or two or more alkynyl groups; and

inducing cycloaddition between the alkynes and azides, thermally or witha suitable catalyst, to covalently crosslink the amphiphiles to eachother near the head groups through formation of triazole groups.

-   16. The method of embodiment 15 wherein the crosslinking agents    comprise two or more azido groups when the polar head groups    comprise alkynyl groups, or two or more alkynyl groups when the    polar head groups comprise azido groups.-   17. The method of embodiment 15 wherein the amphiphiles and water    are in the presence of one or more cargo molecules, wherein the    cargo molecules are thereby encapsulated in the hydrophobic core    upon formation of the self-assembled structure.-   18. The method of embodiment 17 wherein the cargo molecules comprise    one or more drugs, organic nanoparticles, inorganic nanoparticles,    fluorophores, diagnostic agents, catalysts, or a combination    thereof.-   19. The method of embodiment 15 wherein the amphiphiles and water    are in the presence of one or more cargo molecules; the particles    are in the form of vesicles; and the cargo molecules are    encapsulated in water compartments of the vesicles.-   20. The method of any one of embodiments 15-19 further comprising    contacting the surface-crosslinked particle with one or more    azido-containing or alkynyl-containing compounds comprising    water-soluble polymers, fluorophores, biological ligands, nucleic    acids or analogues thereof, or a combination thereof:

inducing cycloaddition between alkynes or azides on the surface of theparticle with the azido-containing or alkynyl-containing compounds,wherein the cycloaddition is induced thermally or with a suitablecatalyst;

to provide a water soluble multivalent particle that has a plurality ofwater-soluble polymers, fluorophores, biological ligands, nucleic acidsor analogues thereof, or a combination thereof, linked to the surface ofthe particle through triazole groups.

-   21. A method for preparing a surface-crosslinked particle    comprising:

combining a plurality of non-polymeric amphiphiles and water to form anoncovalently associated self-assembled structure;

wherein the non-polymeric amphiphiles have polar head groups andnon-polar tail groups, and the polar head groups comprise two or morealkenyl groups;

combining the self-assembled structure with a plurality of crosslinkingagents, wherein the crosslinking agents comprise two or more thiolgroups; and

inducing thiol-ene addition between the alkenes of the amphiphiles andthe thiol groups of the crosslinkers photochemically to covalentlycrosslink the amphiphiles to each other near the head groups through theformation of thioether groups.

-   22. The method of embodiment 21 wherein the amphiphiles and water    are in the presence of one or more cargo molecules; and the cargo    molecules are encapsulated in the hydrophobic core upon formation of    the self-assembled structure.-   23. The method of embodiment 21 wherein the amphiphiles and water    are in the presence of one or more cargo molecules; the particles    are in the form of vesicles; and the cargo molecules are    encapsulated in water compartments formed within the vesicles.-   24. The method embodiment 22 or 23 wherein the cargo molecules    comprise one or more of drugs, organic nanoparticles, inorganic    nanoparticles, fluorophores, diagnostic agents, and catalysts.-   25. The method of any one of embodiments 21-24 further comprising    contacting the surface-crosslinked particle with one or more    thiol-containing compounds comprising water-soluble polymers,    fluorophores, biological ligands, nucleic acids or analogues, or a    combination thereof;

inducing thiol-ene addition reactions between the thiol groups of thethiol-containing compounds and alkene groups at the surface of thesurface-crosslinked particle;

to provide a water soluble multivalent particle that has a plurality offunctional group compounds linked to the surface of the particle throughthioether groups.

-   26. A method for preparing an organic particle comprising:

combining a plurality of nonpolymeric amphiphiles, water, and one ormore nonpolar organic solvents, wherein the amphiphiles comprise one ormore (C₆-C₂₂) alkyl or fluoroalkyl chains and one or more polar headgroups, to provide a noncovalently associated self-assembled structure;

wherein the amphiphiles comprise two or more alkenyl groups near thehead group of the amphiphile, the alkyl or fluoroalkyl chains of theamphiphiles are oriented on the exterior of the self-assembledstructure, and the polar head groups are oriented toward the interior ofthe self-assembled structure; and

irradiating the self-assembled structures, in the presence of aplurality of crosslinking agents comprising two or more thiol groups,and a photoinitiator, to induce crosslinking at the interior of thestructure;

to provide an organic particle comprising amphiphilic moietiescrosslinked by thioether groups.

-   27. The method of any one of embodiments 15, 21, or 26, wherein the    surface crosslinking is cleavable by heat, by a change in pH, by a    reducing agent, or by a combination thereof.-   28. A method of forming a metal nanoparticle comprising contacting a    metal salt and a plurality of particles of embodiment 12 in an    aqueous/organic solvent mixture, thereby extracting metal ions of    the metal salt into the organic solvent, wherein the metal ions    migrate to the interior of the particle, to provide a crosslinked    organic particle encapsulating metal ions; and

contacting the crosslinked organic particle encapsulating metal ionswith a reducing agent, thereby reducing the metal ions in the interiorof the crosslinked organic particle, to provide the metal nanoparticle.

-   29. The method of embodiment 28 wherein the metal salt comprises    AuCl₄ ⁻, PtCl₆ ²⁻, PdCl₄ ²⁻, or a combination thereof.-   30. The method of embodiment 29 wherein more than one type of metal    salt is contacted with the crosslinked organic particle and the    metal nanoparticle formed is an alloy.-   31. A therapeutic method comprising administering to a patient in    need therapy an effective amount of the delivery system of    embodiment 11, wherein the surface crosslinking of the particles    encapsulate one or more drugs, the surface crosslinking of the    particles is cleaved in vivo, and the drug of the particles is    released into the body of the patient, thereby providing the drug to    the patient.-   32. The method of any one of embodiments 15-25 wherein one or more    of the non-polar alkyl or fluoroalkyl groups at the interior of the    particle are chemically degraded, shortened, or removed.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, these embodiments and examplesare only illustrative and do not limit the scope of the invention.Changes and modifications can be made in accordance with ordinary skillin the art without departing from the invention in its broader aspectsas defined in the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. An organic particle comprising a plurality ofnon-polymeric crosslinked amphiphiles; wherein the non-polymericcrosslinked amphiphile comprises one or more nonpolar (C₆-C₆₀)alkyl or(C₆-C₅₀)fluoroalkyl chains and one or more polar head groups; thenonpolar chains are located on the exterior of the particle and thepolar head groups are oriented toward the interior of the particle; andthe amphiphiles are covalently crosslinked to each other near the headgroups through thioether groups comprising an ammonium group bondedthereto.
 2. The organic particle of claim 1, wherein the particlecomprises one or more metal salts or metal particles within the organicparticle.
 3. The organic particle of claim 2, wherein the metal saltcomprises AuCl₄ ⁻, PtCl₆ ²⁻, PdCl₄ ²⁻, or a combination thereof.
 4. Theorganic particle of claim 1, wherein the particle comprises one or morecatalytically active groups oriented toward the interior of theparticle.
 5. The organic particle of claim 4, wherein the catalyticallyactive groups are chosen from a carboxylic acid, sulfonic acid, amine,thiol, or a combination thereof.
 6. The organic particle of claim 1,comprising about 10 to about 150 of the non-polymeric crosslinkedamphiphiles.
 7. The organic particle of claim 1, wherein the non-polartail groups comprise an ester, imine, boronate, disulfide, salt bridge,or a combination thereof linking a portion of the tail to a head groupof the amphiphile or another portion of the tail that is linked to thehead group of the amphiphile.
 8. The organic particle of claim 1,wherein the non-polar tail groups of the amphiphile comprises one ormore (C₆-C₂₂)alkyl groups, (C₆-C₂₂)fluoroalkyl groups, or a combinationthereof.
 9. The organic particle of claim 1, wherein the particle is inthe form of a liposome comprising a bilayer of the amphiphiles, and thebilayer comprises one or more water compartments between the bilayer ofthe amphiphiles.
 10. The organic particle of claim 1, wherein theparticle comprises one or more cargo molecules within the particle or atthe surface of the particle.
 11. The organic particle of claim 10,wherein the cargo molecules comprise one or more of a drug, an organicnanoparticle, an inorganic nanoparticle, a fluorophore, a diagnosticagent, and a catalyst.
 12. The organic particle of claim 1, wherein thecrosslinking is cleavable by heat, by a change in pH, by a reducingagent, or by a combination thereof.
 13. A delivery system comprising aplurality of the organic particles of claim 1 and a pharmaceuticallyacceptable diluent or carrier.
 14. A therapeutic method comprising:administering to a patient in need of therapy an effective amount of thedelivery system of claim 13, wherein the crosslinking of the particlesencapsulates one or more drugs, such that the crosslinking of theparticles is cleaved in vivo and the drug is thereby released into thebody of the patient.
 15. A method for preparing the organic particlecomprising the crosslinked non-polymeric organic amphiphiles of claim 1,the method comprising: combining a plurality of non-polymericamphiphiles, water, and an organic solvent, to form anoncovalently-associated self-assembled structure, wherein thenon-polymeric amphiphile comprises a polar head group and a non-polartail group; combining the self-assembled structure with a crosslinkingagent, wherein a) the crosslinking agent comprises two or more thiolgroups and the polar head group of the non-polymeric amphiphilecomprises two or more alkenyl groups, b) the crosslinking agentcomprises two or more alkenyl groups and the polar head group of thenon-polymeric amphiphile comprises two or more thiol groups, inducingthiol-ene addition between the alkenyl groups and the thiol groups tocovalently crosslink the amphiphiles to each other near the head groupsthrough the formation of thioether groups to form the organic particle.16. The method of claim 15, wherein the crosslinking agent comprises twoor more thiol groups and the polar head group of the non-polymericamphiphile comprises two or more alkenyl groups.
 17. The method of claim15, wherein the crosslinking agent comprises two thiol groups and thepolar head group of the non-polymeric amphiphile comprises atriallylammonium group.
 18. The method of claim 15, wherein theamphiphiles are in the presence of one or more cargo molecules, and thecargo molecules are encapsulated in the hydrophilic core upon formationof the self-assembled structure.
 19. A method of forming a metalnanoparticle comprising: contacting a metal salt and a plurality ofparticles of claim 1 in an aqueous/organic solvent mixture, therebyextracting metal ions of the metal salt into the organic solvent,wherein the metal ions migrate to the interior of the particle, toprovide a crosslinked organic particle encapsulating metal ions; andcontacting the crosslinked organic particle encapsulating metal ionswith a reducing agent, thereby reducing the metal ions in the interiorof the crosslinked organic particle, to provide the metal nanoparticle.20. The method of claim 19, wherein more than one type of metal salt iscontacted with the crosslinked organic particle and the metalnanoparticle formed is an alloy.
 21. The organic particle of claim 1,wherein the ammonium group has the structure: